SPRi cell sensor, preparation method and application thereof

Abstract

The application discloses an SPRi cell sensor and a preparation method and application thereof, wherein the preparation method comprises the following steps: placing a gold film sensing chip in a GSH solution for incubation to obtain a functionalized sensing chip; coupling the functionalized sensing chip to a prism through a refractive index matching liquid, and pressing a PDMS sample flow cell to the surface of the functionalized sensing chip through the pressure of a spring to form a sample detection channel, injecting a nanoantibody fusion protein GST-NB2 into the sample detection channel, and making the nanoantibody fusion protein GST-NB2 pass through the surface of the functionalized sensing chip which is fixed; injecting a BSA solution into the sample detection channel for non-specific site blocking treatment, and then injecting a PBS buffer to remove redundant BSA, thereby obtaining the SPRi cell sensor. The SPRi cell sensor provided by the application can directly inject a living cell suspension within 30 min, and can label-free, quickly and accurately distinguish a HER2 positive cell line and a HER2 negative cell line, and the detection lower limit is 1x10 5 cell / mL of cell concentration.

Description

Technical Field

[0001] This invention relates to the field of cell sensor technology, and in particular to an SPRi cell sensor, its preparation method, and its application. Background Technology

[0002] Overexpression of human surface growth factor receptor 2 (HER2) is a key prognostic factor for breast cancer, with approximately 20-30% of breast cancer patients exhibiting HER2 overexpression. This overexpression is associated with more aggressive cancer cells and shorter survival, making HER2 a crucial indicator for treatment regimens and breast cancer immunotherapy. Over the past decade, targeted therapies against HER2 have been developed, including monoclonal antibodies such as trastuzumab and pertuzumab. These drugs inhibit the proliferation of cells overexpressing HER2 and are effective treatments for HER2-positive cancers. When used in combination with conventional therapies such as chemotherapy, they can significantly improve the treatment outcomes of breast cancer. However, targeted therapies like trastuzumab are expensive, and overuse can lead to cardiotoxicity. Therefore, accurate and rapid monitoring of each patient's cancer cell HER2 status before administering targeted therapy is crucial for finding the optimal dosage and regimen for HER2-positive cancer patients, ultimately maximizing treatment efficacy. Furthermore, HER2 is an important tumor marker associated with many malignancies, making HER2 status detection essential for tumor diagnosis and treatment.

[0003] Early diagnosis and treatment have become the main means of cancer prevention and treatment, necessitating the development of low-cost, high-sensitivity, high-throughput, and rapid technologies and devices for early tumor detection. Utilizing SPRi biosensing technology for label-free detection of interactions between specific receptors and ligands on cell surfaces offers several advantages. First, SPRi eliminates the need for labeled antibodies, which can interfere with their activity, thus more accurately reflecting the natural state of antigen-antibody interactions. Second, SPRi allows real-time monitoring of signal changes induced by cell stimulation without interrupting measurements or collecting delayed signals, unlike flow cytometry. Furthermore, there is a growing societal demand for high-throughput multiplex cell analysis due to the complexity of diseases, such as cancers involving patterns of cell surface antigen expression. While traditional techniques such as fluorescence microscopy and flow cytometry can be used for multiplex analysis, they are time-consuming and limited by fluorescent dyes and filter channels. In contrast, SPRi's sensor surface is easily expandable, allowing for the simultaneous detection of multiple samples.

[0004] The HER2 receptor is a transmembrane protein. HER2-overexpressing (positive) cells exhibit HER2 receptor protein levels tens to hundreds of times higher than surrounding normal breast cells. Clinically, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are commonly used to determine whether cancer cells have high levels of HER2 protein. FISH is considered the "gold standard" for HER2 detection; however, because FISH is expensive and time-consuming, IHC testing is usually performed first. According to the testing criteria, if the IHC result is 0 or 1+, the cancer is considered HER2-negative; if the IHC result is 3+, the cancer is HER2-positive; if the IHC result is 2+, the HER2 status of the tumor is unclear, and further FISH testing of HER2 expression status is needed to clarify the results. However, both methods are limited by several factors, such as expensive antibody labeling tests, long turnaround times, limited analytical sensitivity and target specificity, lack of multiplexing capabilities, and the need for professional operation. These factors significantly impact the reliability of HER2 detection results, making accurate and rapid assessment of HER2 levels and status a challenge for the treatment of HER2-positive cancers. Therefore, developing new HER2 detection methods is of significant clinical importance. Existing technologies still require improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an SPRi cell sensor, its preparation method and application, which aims to solve the problem of low detection sensitivity in existing methods for detecting HER2 in breast cancer cells.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing an SPRi cell sensor, comprising the following steps:

[0008] The cleaned gold film sensor chip was placed in a GSH solution for incubation, so that GSH was modified onto the surface of the gold film sensor chip to obtain a functionalized sensor chip.

[0009] GST-NB2, a nanobody fusion protein that specifically recognizes the extracellular domain of the HER2 receptor, was prepared using prokaryotic expression biology methods.

[0010] The functionalized sensor chip is coupled to a prism via a refractive index matching liquid, and a PDMS sample flow cell is pressed onto the surface of the functionalized sensor chip by a spring to form a sample detection channel. After injecting PBS buffer into the sample detection channel to obtain a stable baseline, the nanobody fusion protein GST-NB2 is injected, so that the nanobody fusion protein GST-NB2 is fixed on the surface of the functionalized sensor chip by directional connection with GSH. Finally, PBS buffer is injected to remove unbound nanobody fusion protein GST-NB2.

[0011] BSA solution was injected into the sample detection channel for non-specific site blocking, and then PBS buffer was injected to remove excess BSA, thus obtaining the SPRi cell sensor.

[0012] The method for preparing the SPRi cell sensor includes incubating a cleaned gold film sensor chip in a GSH solution to modify the surface of the gold film sensor chip with GSH, thereby obtaining a functionalized sensor chip, comprising:

[0013] The initial gold film sensor chip was immersed in acetone solution for ultrasonic cleaning, and then cleaned twice with anhydrous ethanol to remove oily contaminants.

[0014] Next, the initial gold film sensor chip was immersed in NaCl and NaOH solutions to remove the contaminants electrostatically adsorbed on the surface, and then washed twice with ultrapure water and anhydrous ethanol to obtain the gold film sensor chip.

[0015] The cleaned gold film sensor chip was placed in GSH solution and incubated at room temperature for 3-5 hours. Then it was washed twice with DMSO and anhydrous ethanol respectively to obtain the functionalized sensor chip.

[0016] The method for preparing the SPRi cell sensor includes a GSH solution comprising a DMSO solvent and a GSH molecular peptide dispersed in the DMSO solvent, wherein the GSH molecular peptide is composed of glutamic acid, cysteine ​​and glycine.

[0017] The method for preparing the SPRi cell sensor, wherein the preparation of the PDMS sample flow cell includes:

[0018] Design the shape of the PMDS sample cell mold, and then use computer numerical control to process the PMDS sample flow cell mold;

[0019] Pour the prepared polydimethylsiloxane adhesive into the PMDS sample flow cell mold and cure it in a constant temperature oven at 100-200℃ for 15-20 hours to complete the preparation of the initial PDMS sample flow cell.

[0020] A PDMS sample flow cell is prepared by punching holes at both ends of the channel of the initial PDMS sample flow cell using a PDMS puncher, inserting a stainless steel thin tube, and then connecting a silicone microtube through the stainless steel thin tube.

[0021] The SPRi cell sensor is prepared by injecting PBS buffer into the sample detection channel at a flow rate of 10 μL / min for 10 min to obtain a stable baseline. Then, 100 μg / mL of the nanobody fusion protein GST-NB2 is injected at a flow rate of 10 μL / min, so that the nanobody fusion protein GST-NB2 is fixed on the surface of the functionalized sensor chip by directional linking with GSH. After 20 min, PBS buffer is injected to remove unbound nanobody fusion protein GST-NB2.

[0022] The SPRi cell sensor is prepared by injecting 1% BSA solution into the sample detection channel at a flow rate of 10 μL / min for non-specific site blocking, followed by injecting PBS buffer for 30 min to remove excess BSA, thereby obtaining the SPRi cell sensor.

[0023] An SPRi cell sensor, wherein the SPRi cell sensor is prepared using the preparation method of the present invention.

[0024] An application of an SPRi cell sensor, wherein the SPRi cell sensor described in this invention is used to build an intensity-type SPRi sensing system.

[0025] The application of the SPRi cell sensor, wherein the intensity-type SPRi sensing system includes a halogen lamp, and a collimating lens L1, a narrow-band filter F1, a polarizer P1, an SPRi cell sensor, an analyzer P2, a zoom imaging lens L2, and a complementary metal-oxide-semiconductor, arranged sequentially according to the light propagation order.

[0026] Beneficial effects:

[0027] The SPRi cell sensor provided by this invention can, within 30 minutes, rapidly and accurately distinguish between HER2-positive and HER2-negative cell lines without labeling, despite differences in refractive index, size, and adhesion speed among different cells. The detection limit is 1×10⁻⁶. 5 Cell concentration per cell / mL. Attached Figure Description

[0028] Figure 1 This is a flowchart of a method for preparing an SPRi cell sensor according to the present invention.

[0029] Figure 2This is a schematic diagram illustrating the principle of antibody fixation and cell capture in this invention.

[0030] Figure 3 A in the diagram is the optical path and overall architecture of the SPRi system, and B is a physical diagram of the SPRi system.

[0031] Figure 4 The graph shows the test results of the refractive index sensitivity of the intensity-type SPRi sensing system of this invention.

[0032] Figure 5 In the middle, AC are two-dimensional AFM images of bare gold membrane, GSH-modified gold membrane, and antibody-modified gold membrane, respectively. DF are three-dimensional morphology images of bare gold membrane, GSH-modified gold membrane, and antibody-modified gold membrane, respectively.

[0033] Figure 6 A represents the online modification process and response of SPRi for sensor chip and cell capture; B represents the SPR intensity changes at different concentrations of GST-NB2; C represents the SPR intensity changes at different concentrations of GST-NB2 after BSA modification; D represents the real-time SPR response of MDA-MB-231 / EV and MDA-MB-231 / HER2 cells captured at different GST-NB2 concentrations; E represents the SPR intensity changes before and after MDA-MB-231 / EV and MDA-MB-231 / HER2 cells captured at different GST-NB2 concentrations.

[0034] Figure 7 A in the figure shows the monitoring of SPR intensity changes during cell dynamic capture; B shows the SPR intensity changes of four cell lines.

[0035] Figure 8 Optical microscopic images of four cell lines captured on the surface of the sensor chip (scale bar: 200 μm), with the sample channel located in the middle of the white dashed line.

[0036] Figure 9 This is a graph showing the results of SPRi-based detection of HER2 expression in cells.

[0037] Figure 10 This is a graph showing the results of HER2 expression detection in cells based on flow cytometry.

[0038] Figure 11 Figure A shows the signal changes of SPRi when detecting cells at different concentrations. Figure B shows the signal changes when the cell concentration is 1×10⁻⁶. 5 The difference in SPRi signal changes between HER2-negative (HER2-) and HER2-positive (HER2+) cells (N=3, two-way ANOVA test, "***": p<0.001, ns: not significant). Detailed Implementation

[0039] This invention provides an SPRi cell sensor, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0041] This invention provides a method for preparing an SPRi cell sensor, such as... Figure 1 As shown, it includes the following steps:

[0042] S10. The cleaned gold film sensor chip is placed in a GSH solution for incubation, so that GSH is modified onto the surface of the gold film sensor chip to obtain a functionalized sensor chip.

[0043] S20. A nanobody fusion protein, GST-NB2, that specifically recognizes the extracellular domain of the HER2 receptor was prepared using prokaryotic expression biology methods.

[0044] S30. The functionalized sensor chip is coupled to the prism through a refractive index matching liquid, and the PDMS sample flow cell is pressed onto the surface of the functionalized sensor chip by the pressure of the spring to form a sample detection channel. After injecting PBS buffer into the sample detection channel to obtain a stable baseline, the nano-antibody fusion protein GST-NB2 is injected, so that the nano-antibody fusion protein GST-NB2 is fixed on the surface of the functionalized sensor chip by directional connection with GSH. Finally, PBS buffer is injected to remove the unbound nano-antibody fusion protein GST-NB2.

[0045] S40. Inject BSA solution into the sample detection channel for non-specific site blocking treatment, and then inject PBS buffer to remove excess BSA to obtain the SPRi cell sensor.

[0046] In this embodiment, HER2 is an important molecular biomarker for breast cancer treatment and diagnosis. However, the main clinical detection methods are still based on IHC and FISH, which have drawbacks such as low automation, long detection time, large human error, and a high risk of false positives. This embodiment is the first to develop a method for detecting HER2-positive cells based on the SPRi sensing system. With the SPRi cell sensor properly prepared, cell disruption and fluorescence staining are not required. It can rapidly distinguish HER2-overexpressing cells within 30 minutes, and its detection limit is 1×10⁻⁶. 5 Cell concentration at cell / mL has advantages such as fast detection speed, label-free, high throughput, and simple operation. Combined with tumor tissue dissociation technology, it has great potential for clinical application.

[0047] Specifically, most current biosensors rely on immunoglobulin antibodies, but they suffer from limitations such as low stability, high cost, and batch-to-batch variability. Although nanobody technology has made significant progress, research on immobilizing nanobodies on sensing platforms is scarce.

[0048] This invention utilizes a self-prepared HER2 nanobody NB2 (Nanobody 2) as a ligand for cell capture, enabling the capture and detection of live cancer cells using engineered nanobodies on an SPRi sensing system. Nanobodies possess advantages such as high pH stability, strong specificity and affinity, and ease of large-scale production, making them highly advantageous as ligands for SPRi in specific biosensing. These advantages include improved sensor stability and sensitivity, and reduced detection costs. Furthermore, traditional immunoglobulin antibodies (150kDa) are several times larger than nanobodies, while the detection range of SPRi sensors is limited, typically within a depth of 300-500 nm. Therefore, theoretically, a single layer of nanobody modified with an SPRi sensing chip occupies less detection space than a single layer modified with immunoglobulin antibodies, reserving more interaction space for the analyte and achieving a higher ligand density, thereby improving the detection sensitivity of the SPRi for the analyte.

[0049] Immobilizing antibodies on the surface of the gold membrane sensor chip is a crucial step in enabling cell detection in the SPRi system. The quantity, bioactivity, binding site orientation, and stability of the antibody on the gold membrane sensor chip significantly influence its antigen capture ability. Therefore, selecting a suitable immobilization method is extremely important.

[0050] Traditional antibody immobilization methods mainly include physical adsorption, covalent coupling, and affinity coupling. Physical adsorption uses intermolecular forces such as electrostatic adsorption and hydrophobic interactions to immobilize antibodies on a specific substrate surface. This method is very simple, and its advantage is that it does not require chemical treatment of the chip surface and the antibody. However, its disadvantages are obvious: firstly, hydrophobic interactions can, to some extent, alter the spatial structure of the antibody or even inactivate it; secondly, these physical interactions are not as strong as chemical bonds and are easily detached during subsequent processing. The physical adsorption process is random, and antibodies will adsorb in different orientations, reducing the antigen binding efficiency. Covalent coupling is currently the most widely used coupling method, mainly by modifying the solid-phase surface with carboxyl groups and then coupling through the amino groups of the antibody. Since amino groups are ubiquitous on the antibody surface, this coupling method is also random and easily leads to antibody inactivation. Affinity coupling is the most ideal coupling method. Its advantages are that it can well preserve the antibody's activity and achieve directional connection, which can improve the antigen-antibody binding efficiency. Therefore, this invention chose the affinity coupling method.

[0051] Generally, after purifying antibodies using a GST fusion protein expression system, the GST tag is cleaved using enzymes to obtain a single antibody. In this invention, the GST tag is retained, and then the GST-NB2 fusion protein antibody is directionally immobilized by utilizing the affinity between GST and reduced glutathione (GSH). The basic principle is as follows: Figure 2 As shown.

[0052] GSH is composed of glutamic acid, cysteine, and glycine, with the thiol group (-SH) on the cysteine ​​residue being its active group. This invention utilizes the reaction of the thiol group of GSH with gold atoms on a gold surface to form Au-S bonds, thereby modifying the gold surface with GSH. Then, the GST-NB2 fusion nanobody is injected into the gold surface for coupling. The advantages of this modification method are that the affinity coupling between the GST-tagged protein and GSH reduces damage to the activity of the NB2 nanobody, and it can form directional linkages, improving antigen capture efficiency.

[0053] In some specific embodiments, the fabrication of the SPRi cell sensor includes the following steps:

[0054] 1. Cleaning: The initial gold film sensor chip was immersed in acetone solution for ultrasonic cleaning for 20 minutes, and then cleaned twice with anhydrous ethanol to remove oily contaminants. Next, the initial gold film sensor chip was immersed in NaCl and NaOH solutions to remove contaminants adsorbed by electrostatics on the surface. It was then cleaned twice with ultrapure water and anhydrous ethanol, and dried with clean nitrogen gas to obtain the gold film sensor chip.

[0055] 2. Functionalization: The cleaned gold film sensor chip is placed in GSH solution (dissolved in DMSO) and incubated at room temperature for 3-5 hours. Then it is washed twice with DMSO and anhydrous ethanol respectively to obtain the functionalized sensor chip.

[0056] 3. Preparation of PDMS sample flow cell: Design the shape of the PMDS sample cell mold, and then use computer numerical control to process the PMDS sample flow cell mold; pour the prepared polydimethylsiloxane resin into the PMDS sample flow cell mold, and cure it in a constant temperature oven at 100-200℃ for 15-20 hours to complete the preparation of the initial PDMS sample flow cell; use a PDMS punch to punch holes at both ends of the channel of the initial PDMS sample flow cell, insert stainless steel thin tubes, and then connect silicone microtubes through the stainless steel thin tubes to obtain the PDMS sample flow cell.

[0057] 4. Fabrication of the Prism-Coupled SPR Sensing Module: The prism-coupled SPR sensing module includes a prism, a gold film sensing chip, and a sample flow cell. To enable the prism to be reused and reduce manufacturing costs, inexpensive BK7 glass (refractive index 1.515) was selected as the prism material, and the prism size is an equilateral prism of 18mm*18mm. For the fabrication of the sensing chip, a gold film with a thickness of 47nm was deposited on a 1mm*18mm*18mm glass substrate made of BK7 glass using magnetron sputtering. Due to the poor adhesion between the gold film and the glass, it is easy to detach, so a 2nm cadmium (Cr) layer was deposited between the gold film and the glass substrate to increase the stability of the gold film. In use, the sensing chip is coupled to the prism surface using a refractive index matching solution (refractive index 1.518) to reduce light scattering. The sensor chip is bonded to the prism surface using refractive index matching oil, and then the PDMS sample flow cell is pressed onto the sensor chip surface by the pressure of a spring to form a sample detection channel.

[0058] 5. Immobilization: After injecting PBS buffer into the sample detection channel at a flow rate of 10 μL / min for 10 min to obtain a stable baseline, inject 100 μg / mL of the nanobody fusion protein GST-NB2 at a flow rate of 10 μL / min to immobilize the nanobody fusion protein GST-NB2 on the surface of the functionalized sensor chip by directional linking with GSH. After 20 min, inject PBS buffer to remove unbound nanobody fusion protein GST-NB2.

[0059] 6. Blocking: Inject 1% BSA solution into the sample detection channel at a flow rate of 10 μL / min to block non-specific sites, and then inject PBS buffer for 30 min to remove excess BSA to obtain the SPRi cell sensor.

[0060] In some embodiments, an SPRi cell sensor is also provided, which is prepared using the SPRi cell sensor preparation method described in this invention.

[0061] In some embodiments, an application of the SPRi cell sensor is also provided, wherein the SPRi cell sensor described in this invention is used to build an intensity-based SPRi sensing system. In this embodiment, as... Figure 3 As shown, the intensity-type SPRi sensing system includes a halogen lamp, and, in sequence according to the light propagation order, a collimating lens L1, a narrowband filter F1, a polarizer P1, an SPRi cell sensor, an analyzer P2, a zoom imaging lens L2, and a complementary metal-oxide-semiconductor (CMOS). This intensity-type SPRi sensing system uses a halogen lamp (Philips 21V / 150W) as the light source. The white light generated by the halogen lamp is introduced into the SPRi optical path system through a multimode optical fiber, then passes through the collimating lens L1 and the narrowband filter F1 to become monochromatic parallel light. Next, it passes through the polarizer P1 to become P-polarized light (since S-polarized light does not produce the SPR effect, it is filtered out as stray light). The P-polarized light is incident on the SPRi cell sensor at a certain angle, generating the SPR effect. Finally, the reflected light passes through the analyzer P2 and the zoom imaging lens L2 and is received by the complementary metal-oxide-semiconductor (CMOS) for imaging.

[0062] Because sodium chloride has relatively stable physicochemical properties, sodium chloride solutions are commonly used to test the refractive index sensitivity of the intensity-type SPRi sensing system of this invention. This embodiment tested sodium chloride solutions with concentration gradients from 0% to 15%. At room temperature, sodium chloride solutions of 0% (18MΩ pure water), 2%, 4%, 6%, 8%, 10%, and 15% were prepared using ultrapure water. The refractive indices of these solutions were measured using a TDR095 refractive index meter, yielding values ​​of 1.3337, 1.3373, 1.3405, 1.3435, 1.3470, 1.3499, and 1.3582, respectively. Three Regions of Interest (ROIs) were selected for each channel, and sodium chloride solutions were injected sequentially into the four sample channels of the SPRi system in ascending order of concentration. A linear fit was performed on the signal for each ROI, with refractive index units (RIU) as the x-axis and the change in SPR reflected light signal intensity as the y-axis. Calculations showed that the refractive index detection sensitivities of the four channels were 3667 a.u. / RIU, 3821 a.u. / RIU, 3614 a.u. / RIU, and 3605 a.u. / RIU, respectively, with an average sensitivity of S = 3626.75 au / RIU. The detection limit of the sensor for the solution's refractive index can be calculated using the formula to be approximately 1 × 10⁻⁶. -6RIU, therefore the intensity-type SPRi system has excellent detection sensitivity. Meanwhile, from Figure 4 It can be seen that the SPRi system exhibits good linearity in the refractive index solution detection range of 1.3337–1.3499 (Ri). 2 =0.995), when the refractive index of the detected solution reaches 1.3582, the system response is basically outside the linear region, so the dynamic range of the SPRi system is about 0.016RIU.

[0063] Atomic force microscopy (AFM) can detect surface roughness using a cantilever probe, achieving nanometer-level resolution, which is more than a thousand times higher than the optical diffraction limit. It is well-suited for characterizing the morphological effects of biochemical reactions on material surfaces. Therefore, this embodiment used a Bruker Dimension Icon atomic force microscope (AFM) to characterize the morphological changes during the modification of nanobodies onto the sensor chip surface. The morphologies of the bare chip, the GSH-modified chip surface, and the antibody-modified surface were detected using AFM. The images were processed and analyzed using nanoscope analysis software, and the results are as follows: Figure 5 As shown, from left to right, the three gold films are a bare gold film, a gold film modified with GSH, and a gold film modified with an antibody. According to the software's roughness calculation, the root mean square roughness of the three gold films are 0.657 nm, 0.788 nm, and 0.738 nm, respectively, with maximum peak heights of 3.82 nm, 5.33 nm, and 2.85 nm, respectively. The GSH-modified Au-GSH surface is rougher than the bare chip Au surface, while the Au-GSH-NB2 surface is relatively smoother than the Au-GSH surface. Furthermore, from the three-dimensional images, the "peaks" on the Au-GSH surface are smoother than those on the Au-GSH-NB2 surface, indicating that a GST-NB2 antibody layer has formed on the sensor chip surface.

[0064] Antibody concentration optimization results: The injected solution and time during the experiment are as follows Figure 6 As shown in Figure A. Figure 6 Figure B shows the SPR signal response under different concentrations of GST-NB2 fusion nanobody modification. The SPR signal change of GST-NB2 modification increases with increasing concentration, reaching a maximum at 200 μg / mL. However, the cell detection effect is optimal at 100 μg / mL nanobody modification. Figure 6As shown in DE, the reason can be explained by the fact that GST-NB2 can form a stable antibody monolayer on the gold surface within a certain concentration range, so the cell capture rate increases with increasing antibody concentration. However, when the GST-NB2 concentration is too high (200 μg / mL), an unstable multilayer will form on the surface. When cells flow through, they will carry away the unstable GST-NB2 through affinity binding, resulting in signal attenuation. This can be demonstrated by the changes in SPR signal before and after BSA injection for blocking, such as... Figure 6 As shown in Figure C, the higher the antibody concentration, the weaker the blocking effect of BSA. When the antibody concentration increases from 100 μg / mL to 200 μg / mL, the signal change before and after BSA blocking also changes from positive to negative. Therefore, when the antibody concentration is 100 μg / mL, a monolayer of antibody is basically formed on the surface of the sensor chip. Therefore, in this embodiment, a GST-NB2 fusion nanobody with a concentration of 100 μg / mL was selected for modifying the sensor chip.

[0065] To verify that the SPRi cell sensor prepared in this invention can specifically and sensitively detect HER2, the following experimental tests were also conducted in this application:

[0066] 1. Cell selection:

[0067] Cells are the basic structural and functional units of organisms, and are a much more complex research and detection subject than proteins. Different cell lines exhibit significant differences in physical properties such as refractive index and size, as well as biological characteristics such as adhesion and attachment speed. These variables can greatly influence the SPRi signal. Therefore, to control these variables and demonstrate the specificity of the SPRi sensing system for capturing live cells with high HER2 expression, we selected the rapidly proliferating breast cancer cell line MDA-MB-231 and transfected it with a plasmid containing the HER2 expression gene. Then, through screening with an resistance gene, we constructed a stable transfected cell line, MDA-MB-231 / HER2, with exogenous HER2 protein overexpression, and a control cell line, MDA-MB-231 / EV, transfected with an empty vector plasmid. Through this method, we obtained a pair of engineered breast cancer cells that were essentially identical in characteristics except for the difference in HER2 protein expression levels. Furthermore, to make the experiment more generalizable, we also selected the normal breast epithelial cell line MCF-10A and the wild-type HER2-positive breast cancer cell line BT-474 as the subjects for SPRi detection. In summary, the experimental subjects selected in this experiment included MDA-MB-231 / EV, MDA-MB-231 / HER2, MCF-10A, and BT-474 cell lines. Using IHC methods and clinical standards for analysis, the HER2 expression scores of MDA-MB-231, MCF-10A, and BT-474 were 0-1+, 0-1+, and 3+, respectively, indicating that MDA-MB-231 / EV and MCF-10A are HER2-negative cells, while BT-474 is a HER2-positive cell line.

[0068] 2. Preparation of single-cell suspension:

[0069] After two passages, the newly revived cells are prepared into single-cell suspensions once they reach the exponential growth phase (approximately 70% density) and the adherent cells have reached this stage. The method is as follows: First, open the cell culture flask, discard the original culture medium, and wash twice with PBS to remove cell metabolites and residual culture medium, avoiding the influence of serum in the medium on trypsin digestion efficiency. Then, add 1 mL of trypsin to cover the cells and incubate in a CO2 incubator for digestion. Once the cells shrink and become rounded (mostly detached), add culture medium containing serum to stop trypsin digestion. Transfer the cells to centrifuge tubes and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, add PBS, gently resuspend the cells using a pipette, centrifuge again, and resuspend in PBS. Finally, count the cells using a cell counting chamber and dilute to the required experimental concentration.

[0070] Based on experimental experience, the digestion time for MDA-MB-231 / EV and MDA-MB-231 / HER2 was set at 2.5 min, the digestion time for BT-474 was set at 3 min, and the digestion time for MCF-10A cells was set at 12 min.

[0071] 3. SPRi-based HER2-positive cell capture assay:

[0072] After completing the orientation and immobilization of the nanobody on the sensor chip surface and BSA blocking, in this embodiment, the same concentration (2.5 × 10⁻⁶) was continuously injected at a flow rate of 5 μL / min over a period of 30 min. 6 Four cell lines (cells / mL) were used to study cell-specific capture in PBS suspensions. The difference between the SPR signal at 30 min after cell injection and the baseline SPR signal was taken as the SPR signal change for cell detection.

[0073] The process of capturing live cancer cells under continuous flow conditions can be achieved through... Figure 7 The process can be explained by the several stages shown in Figure AB. At the moment of cell injection, the cell concentration is high, and some cells rapidly diffuse to the sensor chip surface, causing a rapid increase in SPR intensity. Subsequently, due to gravity, the cells gradually unfold, with HER2-positive cells being captured by the nanobodies and HER2-negative cells being lost. Flow cytometry results showed that BT-474 cells, being larger and more granular than MDA-MB-231 / HER2 cells, exhibited a faster increase in SPR intensity when capturing the same number of cells. Therefore, at this stage, the SPR intensity of the two cell types begins to diverge. Based on our previous experiments optimizing the flow rate, we found almost no cells detected in the channel 10 minutes after injection. Nevertheless, a continued increase in SPR intensity was still observed. This can be attributed to the diffusion and attachment of live cells to the sensor chip surface. Therefore, more nanobodies can interact with the HER2 ECD, drawing cells into the plasma field and promoting continuous interaction with the nanobodies. Thus, the rate of SPR intensity increase largely depends on the cell attachment properties.

[0074] 1.5 hours after the experiment was terminated, the sensor chip was observed using an inverted optical microscope, such as... Figure 8As shown, most BT-474 cells remained round in the unattached state, while most MDA-MB-231 / HER2 cells were flattened in the attached state. This indicates that the attachment rate of MDA-MB-231 / HER2 is faster than that of BT-474. This observation explains why the SPR intensity change of MDA-MB-231 / HER2 cells is faster than that of BT-474 cells. Therefore, despite being injected with the same cell concentration, different cell lines exhibited different curve trends during cell flow capture, suggesting that the SPRi cell assay method can also reflect cell attachment and growth characteristics, as well as cell refractive index information, to some extent.

[0075] Based on the above analysis, it can be concluded that the specific binding of the overexpressed HER2 receptor to the nanobody on the sensor chip surface, coupled with the attachment of living cells to the surface, resulted in a higher SPR intensity for the HER2-positive cell lines MDA-MB-231 / HER2 and BT-474 within 30 minutes compared to the HER2-negative cell lines MDA-MB-231 / EV and MCF-10A. Figure 9 As shown, this resulted in a 4 to 5-fold change in SPR intensity, with MCF-10A at 1.653, MDA-MB-231 / EV at 2.019, BT-474 at 9.275, and MDA-MB-231 / HER2 at 9.385. To better demonstrate the accuracy of the SPRi cytometry assay for HER2 detection, we performed a comparative analysis with flow cytometry, such as... Figure 10 As shown, signal intensities were observed for MCF-10A (3.904), MDA-MB-231 / EV (4.083), BT-474 (5.654), and MDA-MB-231 / HER2 (6.037). The SPRi detection results and the signal intensity trends of the four cell types were consistent with the flow cytometry results. Notably, the signal difference in HER2-positive cells was several times higher than that in HER2-negative cells, confirming the method's ability to accurately distinguish between HER2-positive and HER2-negative cells. Furthermore, the SPR intensity increase in HER2-negative cells was relatively gradual, indicating good nonspecificity and a high signal-to-noise ratio.

[0076] 4. Detection limit of HER2-positive cells:

[0077] To further test the detection limit of positive HER2 cells in the SPRi system, we separately applied 1×10⁻⁶ cells to the SPRi system. 5 cells / mL, 5×10 5 cells / mL, 1×10 6 cells / mL, 2×10 6Four cell suspensions at different concentrations (cells / mL) were injected into the SPRi system for flow capture experiments. Changes in SPR signals in the four cell types were observed. Figure 11 As shown in Figure A, when the cell concentration is greater than 5 × 10⁻⁶ 5 At a cell concentration of 1×10⁶ cells / mL, the signal-to-noise ratio (SNR) for HER2-positive cells was high. As cell concentration decreased, the difference in SPR signal changes between HER2-positive and HER2-negative cells gradually decreased, leading to a lower SNR. 5 At a rate of cells / mL, the signal-to-noise ratio is low, but the changes in SPR signal values ​​between HER2-positive and HER2-negative cells still show significant differences, such as... Figure 11 As shown in Figure B. Therefore, under the above experimental conditions, the detection limit of the intensity-type SPRi sensing system for HER2-positive cells is approximately 1 × 10⁻⁶. 5 cells / mL.

[0078] In summary, this invention involves the targeted placement of nanobodies on the surface of a sensor chip, and the expression status of the HER2 receptor in four different cell lines (MDA-MB-231 / EV, MDA-MB-231 / HER2, MCF-10A, and BT-474) was analyzed using a specific cell capture method. The results show that, despite the influence of various factors such as cell size, particle size, and adhesion characteristics, the intensity-based SPRi biosensor system can perform label-free, rapid, and accurate identification of HER2-positive and HER2-negative cell lines within 30 minutes of direct injection of a live cell suspension, using low cell sample volume (<200 μL) and concentration (10^5 cells / mL). Compared to IHC, the greatest advantage of the SPRi cell detection method is that it obtains results in a shorter time without the need for fixation and staining, and the operation is more simplified.

[0079] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing a SPRi cell sensor, characterized by, Including the following steps: The cleaned gold film sensor chip was placed in a GSH solution for incubation, so that GSH was modified onto the surface of the gold film sensor chip to obtain a functionalized sensor chip. GST-NB2, a nanobody fusion protein that specifically recognizes the extracellular domain of the HER2 receptor, was prepared using prokaryotic expression biology methods. The functionalized sensor chip is coupled to a prism using a refractive index matching liquid. A PDMS sample flow cell is pressed onto the surface of the functionalized sensor chip by a spring to form a sample detection channel. After injecting PBS buffer into the sample detection channel to obtain a stable baseline, the nanobody fusion protein GST-NB2 is injected. The nanobody fusion protein GST-NB2 is then directionally linked and fixed to the surface of the functionalized sensor chip through the affinity coupling between the GST tag in the nanobody fusion protein GST-NB2 and GSH. Finally, PBS buffer is injected to remove unbound nanobody fusion protein GST-NB2. BSA solution was injected into the sample detection channel for non-specific site blocking, and then PBS buffer was injected to remove excess BSA, thus obtaining the SPRi cell sensor. The concentration of the nanoantibody fusion protein is 20-100 μg / m.

2. The method for preparing the SPRi cell sensor according to claim 1, characterized in that, The cleaned gold film sensor chip is incubated in a GSH solution to allow GSH to be deposited onto the surface of the gold film sensor chip, thus obtaining a functionalized sensor chip, comprising: The initial gold film sensor chip was immersed in acetone solution for ultrasonic cleaning, and then cleaned twice with anhydrous ethanol to remove oily contaminants. Next, the initial gold film sensor chip was immersed in NaCl and NaOH solutions to remove the contaminants electrostatically adsorbed on the surface, and then washed twice with ultrapure water and anhydrous ethanol to obtain the gold film sensor chip. The cleaned gold film sensor chip was placed in GSH solution and incubated at room temperature for 3-5 h. Then it was washed twice with DMSO and anhydrous ethanol respectively to obtain the functionalized sensor chip.

3. The method for preparing the SPRi cell sensor according to claim 1, characterized in that, The GSH solution comprises DMSO solvent and GSH molecular peptides dispersed in DMSO solvent, wherein the GSH molecular peptides are composed of glutamic acid, cysteine ​​and glycine.

4. The method for preparing the SPRi cell sensor according to claim 1, characterized in that, The preparation of the PDMS sample flow cell includes: Design the shape of the PMDS sample cell mold, and then use computer numerical control to process the PMDS sample flow cell mold; Pour the prepared polydimethylsiloxane adhesive into the PMDS sample flow cell mold and cure it in a constant temperature oven at 100-200℃ for 15-20 h to complete the preparation of the initial PDMS sample flow cell. A PDMS sample flow cell is prepared by punching holes at both ends of the channel of the initial PDMS sample flow cell using a PDMS puncher, inserting a stainless steel thin tube, and then connecting a silicone microtube through the stainless steel thin tube.

5. The method for preparing the SPRi cell sensor according to claim 1, characterized in that, After injecting PBS buffer into the sample detection channel at a flow rate of 10 μL / min for 10 min to obtain a stable baseline, 100 µg / mL of the nanobody fusion protein GST-NB2 was injected at a flow rate of 10 μL / min to fix the nanobody fusion protein GST-NB2 on the surface of the functionalized sensor chip by directional linking with GSH. After 20 min, PBS buffer was injected to remove unbound nanobody fusion protein GST-NB2.

6. The method for preparing the SPRi cell sensor according to claim 1, characterized in that, 1% BSA solution was injected into the sample detection channel at a flow rate of 10 μL / min for non-specific site blocking, and then PBS buffer was injected for 30 min to remove excess BSA, thus obtaining the SPRi cell sensor.

7. An SPRi cell sensor, characterized in that, The SPRi cell sensor was prepared using the preparation method described in any one of claims 1-6.

8. An application of an SPRi cell sensor, characterized in that, The SPRi cell sensor described in claim 7 is used to build an intensity-type SPRi sensing system.

9. The application of the SPRi cell sensor according to claim 8, characterized in that, The intensity-type SPRi sensing system includes a halogen lamp, and collimating lens L1, narrowband filter F1, polarizer P1, SPRi cell sensor, analyzer P2, zoom imaging lens L2, and complementary metal-oxide-semiconductor arranged in sequence according to the light propagation order.

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