A highly robust biosensor for in vitro detection of her2 antigen

Abstract

The application relates to a high-robustness biosensor for in-vitro HER2 antigen detection, which comprises a substrate, a dielectric waveguide layer, a periodic dielectric nanopillar array, a HER2 recognition functional layer and a microfluidic clamp. The periodic dielectric nanopillar array is formed by two-dimensional extension of a cell with a planar period of 610 nm, each cell comprising two square-section dielectric nanopillars with the same height, different bottom side lengths and adjacent side surfaces in contact with each other; the dielectric waveguide layer has a thickness of 200 nm, and the bottom side lengths of the two dielectric nanopillars are 183 nm and 122 nm respectively. Through the combination of the structural parameters, the Q factor divergence points corresponding to two symmetric protected continuous domain bound states are close to each other in the momentum space and form a near-merging state, thereby expanding the high-Q factor region near the near-merging state and improving the tolerance of the sensor to structural size deviation and test disturbance.

Description

Technical Field

[0001] This invention relates to the field of optical detection chip technology, and in particular to a highly robust biosensor for in vitro HER2 antigen detection. Background Technology

[0002] With the increasing demands of biomedicine, label-free biosensor technology with ultra-low concentrations has become a key technology in early diagnosis, precision medicine, and drug development. Optical biosensors (OBS) have attracted significant attention due to their advantages such as high sensitivity, resistance to electromagnetic interference, and non-contact reading. In label-free OBS technology, the target analyte exists in its natural form. When a liquid containing the target analyte comes into contact with the sensor, the recognition units on the sensor surface specifically capture the analyte, causing changes in the optical properties of the surface structure (such as refractive index and absorbance), thereby altering the optical response of the device. This technology combines ease of use, low cost, rapid response, and real-time monitoring capabilities. Among numerous optical sensing technologies, biosensors with integrated circuits (BICs) have become an important physical mechanism for the design of high-sensitivity optical sensors in recent years due to their ability to achieve extremely high quality factors (Q factors) and localized enhanced light-matter interactions.

[0003] In recent years, bound confined states (BICs) have provided new opportunities for the design of high-Q optical devices. Optical BICs are special bound states located within the radiation continuum, where radiation is completely suppressed. Based on differences in radiation suppression mechanisms, they are generally classified into symmetric-protected and sporadic types. Ideally, BICs possess an infinitely large Q factor and a highly localized electromagnetic mode field. However, in practical structures, due to factors such as excitation deviations, size limitations, material absorption, and fabrication errors, BICs degenerate into quasi-BICs, with finite Q factors, which places higher demands on their practical fabrication.

[0004] To address these long-standing problems, merging BICs was proposed and implemented. Multiple topological charges are adjusted to be very close to each other, which can expand the momentum range of quasi-BICs with high Q factors and suppress out-of-plane scattering losses caused by structural disorder. The essence of merging BICs is to adjust the wave vector or structural parameters so that multiple originally isolated BICs gradually approach and eventually merge in the parameter space. When two BIC modes are not completely merged (i.e., near-merging), the Q factor, which should have decayed sharply with the system parameters, is "flattened" within a specific parameter range, making the performance of the corresponding mode insensitive to parameter perturbations within that range. This characteristic endows merged BICs with the ability to resist perturbations. Therefore, it has great research value and application potential in realizing enhanced second harmonic generation, lasers, and highly robust optical sensing platforms.

[0005] Despite this, existing designs still face several challenges. Current research on merged BICs largely relies on combinations of symmetric-protected BICs and sporadic BICs. Existing merged BIC structures typically bring multiple originally separate Q-factor divergence points closer together by adjusting the relative positions of different types of BICs in momentum space. For BICs or quasi-BICs located outside the Brillouin zone center, resonance readout usually requires selecting the corresponding in-plane wavevectors through angle resolution or oblique incidence, increasing the complexity of optical path adjustment and device integration. Furthermore, in practical devices with substrates, finite array sizes, and fabrication deviations, the radiation suppression conditions of ideal BICs are compromised, and their high Q characteristics may significantly decrease. Therefore, how to obtain near-merged BICs using fabricable planar dielectric structures and maintain stable measurable resonances under biofunctionalization, liquid flow, and long-term detection conditions remains a technical problem to be solved. Meanwhile, robustness, a key requirement for many optical applications, has not yet been systematically explored in merged BIC research. In addition, most optical sensors based on merged BICs are limited to volume refractive index sensitivity sensing. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, the present invention provides a highly robust biosensor for in vitro HER2 antigen detection.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] A highly robust biosensor for in vitro HER2 antigen detection includes a sensor body; the sensor body includes a substrate layer and a cell array disposed on the substrate layer; the cell array includes multiple cell unit structures; the cell unit structure includes a convex structure formed by closely arranged first square pillars and second square pillars; the first square pillars and the second square pillars have the same height; the side length of the first square pillar is w1, satisfying 180nm < w1 ≤ 250nm; the side length of the second square pillar is w2, satisfying 100nm ≤ w2 ≤ 180nm; after a HER2 recognition functional layer is disposed on the surface of the sensor body, it is integrated with a microfluidic fixture to form a highly robust biosensor for in vitro HER2 antigen detection.

[0009] In one embodiment of the present invention, the substrate layer includes a substrate and a waveguide layer; the cell array is disposed on the waveguide layer.

[0010] In one embodiment of the present invention, the substrate material is silicon, quartz, or glass.

[0011] In one embodiment of the present invention, the height of the waveguide layer is t. w Satisfying 200nm≤t w ≤220nm.

[0012] In one embodiment of the present invention, the t w =200nm.

[0013] In one embodiment of the present invention, a plurality of unit cell structures are periodically arranged on the substrate in a first direction and a second direction, and the arrangement period of the plurality of unit cell structures in the first direction and the second direction is P; the first direction is perpendicular to the second direction; and P is 590-620 nm.

[0014] In one embodiment of the present invention, the material of the unit cell structure is silicon nitride or titanium dioxide dielectric material; the refractive index is 1.8-2.5.

[0015] In one embodiment of the present invention, the heights of the first square column and the second square column are t. g Satisfying 80nm≤t g ≤120nm.

[0016] In one embodiment of the present invention, w1 = 183 nm; w2 = 122 nm; t g =100nm; the arrangement period of the unit cell structure is 610nm.

[0017] In one embodiment of the present invention, the HER2 recognition functional layer is formed sequentially through the following steps: hydroxylation treatment, silanization, immobilization of Protein G, immobilization of HER2 antibody, and BSA blocking.

[0018] The beneficial effects of this invention are: (1) It is fundamentally different from traditional metasurface sensing structures based on a single resonance mode. This invention adopts a dual-symmetric protected BIC merging mechanism to form a dual Q-factor divergence point structure in momentum space. The coupling and merging of two symmetric protected BICs are achieved by adjusting the structural geometric parameters, and the Q-factor distribution is "flattened" within a specific parameter range. This mechanism avoids dependence on sporadic BICs, and while maintaining an ultra-high quality factor, it significantly enhances the structure's tolerance to processing errors and parameter disturbances, thereby improving device stability and repeatability;

[0019] (2) The period of the metasurface structure is 590-620nm, the thickness of the silicon nitride waveguide layer is 100-250nm, the thickness of the pattern layer is 80-120nm, and the widths w1 and w2 of the two square pillars of the "convex" structure are in the range of 100-250nm and w1≠w2. The values ​​of w1 and w2 enable the two symmetric protected BIC modes to merge in momentum space.

[0020] (3) The all-dielectric periodic nanostructure provided by the present invention is a silicon nitride metasurface array structure, which does not require the participation of metal materials and does not have the problem of metal absorption loss. Since the all-dielectric material has low optical loss characteristics, it can support high quality factor resonance mode, and at the same time enhance the local distribution of electromagnetic field on the surface of nanostructure, significantly improve the optical field energy density and field-matter overlap factor, thereby enhancing the sensing sensitivity;

[0021] (4) The dual-symmetric protected merged BIC metasurface structure provided by this invention maintains stable high-Q characteristics within a certain range of geometric parameter perturbations, achieving robust response to small deviations in structural dimensions. Compared with the shortcomings of traditional single BIC or quasi-BIC structures that are extremely sensitive to parameters, this invention makes it easier to obtain high-performance devices under actual processing conditions, reduces fabrication difficulty and cost, and improves device consistency;

[0022] (5) The metasurface chip constructed in this invention is integrated with a microfluidic system. The chip itself serves as an open planar sensing platform that can directly contact liquid samples, enabling real-time transmission and detection of the biological solution to be tested. This structure avoids the complex optical cavity packaging process, simplifies the system structure, improves detection efficiency, and is beneficial for continuous flow detection and dynamic monitoring.

[0023] (6) The merged BIC resonance mode supported by this invention has an extremely high quality factor and significantly enhanced near-field localization capability, so that even a small change in refractive index can cause a significant resonant wavelength shift. By monitoring the changes in the peak position of the reflection spectrum at high resolution, it is possible to respond to extremely weak molecular binding events, thereby achieving highly sensitive detection of low concentrations of HER2 protein;

[0024] (7) This invention employs a surface functionalization strategy of Protein G-directed antibody immobilization and BSA blocking of unbound sites, which significantly improves antibody orientation consistency and antigen binding efficiency, reduces non-specific adsorption, and enhances detection specificity and signal-to-noise ratio. This method ensures the controllability and repeatability of the molecular binding process, providing a reliable basis for high-precision quantitative detection;

[0025] (8) The dual-Q divergence coupling mechanism proposed in this invention maintains a high-Q state within a certain parameter range, enabling the sensor to maintain a stable response even in complex biological environments. This characteristic gives it a significant advantage in low-concentration or even picomolar-level detection, with a detection limit as low as 1 pM, making it suitable for demanding applications such as early screening of tumor markers;

[0026] (9) The sensing and detection system constructed in this invention has a compact structure, simple optical path, and is easy to integrate with conventional spectral detection equipment. The system has low construction cost and simple operation process, and has good engineering application prospects and clinical promotion potential. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the cell structure of the highly robust BICs biosensor of the present invention;

[0029] Figure 2 This is a top view of the cell structure of the highly robust BICs biosensor of the present invention;

[0030] Figure 3 This invention presents the reflectance spectra of the highly robust BICs biosensor under different ff2 conditions;

[0031] Figure 4 This is a schematic diagram of the spatial optical path coupling system used in the detection of the high-robustness BICs biosensor of this invention;

[0032] Figure 5 These are the experimental reflectance spectrum and linewidth fitting spectrum of the highly robust BICs biosensor of this invention;

[0033] Figure 6 These are multiple sets of spectra of the highly robust BICs biosensor of this invention in solutions with different refractive indices;

[0034] Figure 7 This is a robustness test chart (stability test of FWHM over time) of the high robustness BICs biosensor of the present invention.

[0035] Figure 8 This is a detection diagram of the HER2 antigen molecule optical signal of the highly robust BICs biosensor of this invention;

[0036] Figure 9 This is a side view of the surface of the sensor body of the present invention having a HER2 recognition function layer.

[0037] Explanation of reference numerals in the attached figures: 1. Substrate; 2. Waveguide layer; 31. First square pillar; 32. Second square pillar; 4. HER2 recognition functional layer. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0039] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," "front end," "rear end," "both ends," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0040] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0041] A robust biosensor for in vitro HER2 antigen detection employs a convex-shaped unit cell structure with closely arranged first square pillars 31 and second square pillars 32. This unit cell structure forms a unit cell array mounted on a substrate. The difference in the dimensions of the two pillars introduces in-plane symmetry breaking, exciting a merged BIC resonance mode. The optimal parameter ranges obtained through extensive experiments are: the side length w1 of the first square pillar 31 is 180-250 nm, and the side length w2 of the second square pillar 32 is 100-180 nm. This size combination allows the two symmetry-protected BIC modes to achieve a near-merging state in momentum space, thereby expanding the high Q-factor distribution region and improving the sensor's robustness to manufacturing errors. Compared to traditional single-pillar or symmetrical double-pillar structures, this convex-shaped structure can simultaneously achieve high Q-factor and high-field localization effects, laying a structural foundation for subsequent high-sensitivity biological detection.

[0042] In one embodiment of the present invention, the substrate layer consists of a substrate 1 and a waveguide layer 2, with the substrate 1 and waveguide layer 2 stacked together, and a cell array disposed on the waveguide layer 2. This dual-layer structure design can effectively guide the propagation of the light field within the waveguide layer 2, enhancing the interaction length between the light and the cell array. The presence of the waveguide layer 2 can better confine the electromagnetic field energy near the nanostructure, thereby improving the quality factor of the resonant mode. The cell array is directly fabricated on the waveguide layer 2, ensuring the optical continuity of the structure, avoiding the additional losses introduced by the intermediate layer, and facilitating the achievement of higher sensing sensitivity.

[0043] In one embodiment of the present invention, the substrate 1 is made of silicon, quartz, or glass. All three materials have excellent optical transparency and mechanical stability, and extremely low absorption loss in the near-infrared band (the operating wavelength of this sensor is approximately 950 nm).

[0044] In one embodiment of the present invention, the height tw of waveguide layer 2 is in the range of 100-250 nm. This thickness range has been rigorously verified by mode simulation, and the preferred thickness range of 200-220 nm can achieve optimal mode constraint and loss balance, so that the Q factor of the resonant mode reaches the highest level.

[0045] In one embodiment of the invention, the cell unit structure has an arrangement period P of 590-620 nm in two perpendicular directions. This period range allows the resonance wavelength to fall within the near-infrared window of 900-1000 nm, within which biological tissue absorption and scattering are minimized, while also optimizing the performance of commercial spectrometers and light sources. The preferred period of 610 nm, in particular, achieves optimal coupling between the two BIC modes, maximizing the Q-factor flattening region. The first direction is parallel to the X-axis, and the second direction is parallel to the Y-axis.

[0046] In one embodiment of the present invention, the cell unit structure is made of silicon nitride or titanium dioxide with a refractive index of 1.8-2.5. Both of these dielectric materials exhibit extremely low optical loss in the near-infrared band, far superior to metallic materials, and can support high-quality factor resonance. The refractive index range of 1.8-2.5 forms a sufficient refractive index difference with the substrate material (quartz / glass with a refractive index of approximately 1.45), enabling effective confinement of the optical field.

[0047] In one embodiment of the present invention, the height tg of the first square pillar 31 and the second square pillar 32 is in the range of 80-120 nm. This height range is optimized to achieve optimal localization of the light field in the vertical direction, so that most of the electromagnetic field energy is distributed in the sensing region near the surface of the nanopillar, thereby enhancing the interaction intensity between light and the biomolecules to be measured.

[0048] In one embodiment of the present invention, w1=183nm, w2=122nm, tg=100nm, and the arrangement period is 610nm. This parameter combination is the optimal solution obtained through extensive simulation and experimental verification, and can achieve a perfect near-merging state of two symmetric protected BIC modes. Under these parameters, the Q factor has the greatest tolerance for structural size deviations, and the overall performance is optimal.

[0049] In one embodiment of the present invention, the preparation process of the HER2 recognition functional layer adopts a five-step surface functionalization method: hydroxylation treatment introduces active hydroxyl groups on the surface of the dielectric nanopillars, providing reaction sites for subsequent silanization; the silanization step modifies amino groups on the surface using APTES molecules, achieving the transition from the inorganic material surface to the organic functional layer; the Protein G immobilization step utilizes the specific binding of Protein G to the antibody Fc fragment to achieve targeted antibody immobilization, which can improve antibody binding activity by 3-5 times compared to random immobilization methods; the HER2 antibody immobilization step achieves the assembly of specific recognition units; and the BSA blocking step blocks unbound active sites, reduces non-specific adsorption, and improves the signal-to-noise ratio of detection. This functionalization process can ensure the high activity and high specificity of the HER2 antibody, providing a molecular recognition basis for the highly sensitive detection of low-concentration HER2 antigen.

[0050] The highly robust BICs biosensor for ultra-low concentration biological detection provided by this invention has the following structure: Figure 1 As shown, by jointly setting the cell period, dielectric waveguide layer thickness, pattern layer height, and the base side lengths of the two dielectric nanopillars (first square pillar 31 and second square pillar 32), the structure supports the Q-factor divergence points corresponding to two symmetric protected BICs, and the two Q-factor divergence points gradually approach each other in momentum space, forming a near-merged BIC state. Compared with structures that excite isolated quasi-BIC resonances by only changing the in-plane asymmetry, the near-merged state can expand the range of wave vectors or structural parameters corresponding to high Q-factor distributions, reducing the sensitivity of resonance performance to finite size, processing deviations, and test disturbances. For an ideal infinite periodic model, the simulated radiation Q-factor of the structure can reach up to 6.04 × 10⁻⁶. 6 The loading Q factor of the actual device is also affected by material absorption, finite array size, surface roughness, incident beam divergence angle and measurement resolution.

[0051] This invention also solves the problem of how to maintain the measurability and stability of high Q resonance under certain structural deviations and long-term testing conditions with substrate, limited array size, biofunctionalized layer (HER2 recognition functional layer) and microfluidic disturbance.

[0052] To address the aforementioned issues, this application does not modify a single geometric dimension in isolation, but rather adjusts the cell period P and the dielectric waveguide layer thickness t. w Pattern layer height t g The base side lengths w1 and w2 of the two dielectric nanopillars are set as an interrelated parameter combination. This parameter combination causes the Q-factor divergence points corresponding to the two symmetry-protected BICs to be in a near-merging state in momentum space, thereby expanding the high Q-factor region near the near-merging state.

[0053] Therefore, the technical effect obtained by this application is not a simple increase in the isolated quasi-BIC resonance Q factor, nor is it a general shift in the positions of the two spectral peaks. Rather, it is a change in the distribution range of the high Q factor in the wave vector space or structural parameter space, which reduces the sensitivity of the device to finite size, processing errors and test disturbances.

[0054] This invention provides a method for detecting HER2 protein based on the aforementioned periodic nanostructure, comprising the following steps: The cleaned sample is sequentially ultrasonically cleaned with acetone, ethanol, and deionized water, and then dried to remove organic residues such as photoresist. The sample is then immersed in a freshly prepared piranha solution to remove organic contaminants and introduce hydroxyl groups onto the surface at room temperature. After removal, the sample is repeatedly rinsed with deionized water and ultrasonically cleaned three times, dried, and then treated with oxygen plasma to increase hydroxyl density. Next, the sample is placed in an APTES / ethanol solution to modify the surface with amino groups, followed by cleaning with anhydrous ethanol and drying. Protein G solution is spotted onto the modified sensor surface, allowed to stand in a constant humidity environment, sealed overnight, and then installed in a microfluidic channel system. Unbound Protein G is washed with PBS buffer. Subsequently, HER2 antibody solution is spotted at the same location, again allowed to stand, sealed overnight, and then washed with PBS to remove unbound antibody. Finally, BSA solution is introduced to block non-specific binding sites, and the sample is washed with PBS until the baseline stabilizes, completing the surface functionalization modification. Finally, different concentrations of HER2 antigen solution were sequentially introduced for testing, followed by washing with PBS to eliminate volume refractive index interference, and the resonance peak shift was monitored in real time.

[0055] Example 1:

[0056] On substrate 1, there is a waveguide layer 2 with a thickness tw of 200 nm. On this waveguide layer 2 is the designed metasurface array structure (cell array). The cell array height tg is 100 nm, the array period P is 610 nm, the width of the first square pillar 31 is w1, and the width of the second square pillar 32 is w2. See [reference needed]. Figure 1-2 The quasi-BICs pattern is generated by adjusting the width of adjacent square columns and making w1≠w2 to achieve symmetry breaking within the structure.

[0057] Figure 3This indicates that altering the in-plane geometric asymmetry can control the radiative coupling and spectral visibility of the two quasi-BIC resonances. When ff2 = 0 (complete structural symmetry), the BIC modes do not radiate energy and are invisible in the spectrum; the bright state excited at 957 nm should be classified as GMR. When ff2 = 0.4, the in-plane C2 symmetry of the structure is broken. This symmetry breaking causes radiative leakage in the two BIC modes, leading to the excitation of two extremely sharp qBIC resonances, qBIC1 and qBIC2, at 951 nm and 964 nm, respectively. When ff2 further increases to 0.6, the structural symmetry is restored, and the two qBIC modes are once again protected by symmetry, no longer coupling with the radiation field and thus disappearing from the spectrum. Simultaneously, with the increase of ff2, the effective refractive index also increases, causing a certain degree of redshift in the GMR modes. Furthermore, by demonstrating the existence of two divergences in the Q factor, the Q factor of the qBIC mode changes smoothly within a certain parameter range. Within the study interval, regardless of the value of ff1, the qBIC mode can always recover to the ideal symmetry protection state at ff2=0 and ff2=ff1. Therefore, by simply manipulating the value of ff1, the position of the symmetry protection state excited at ff1=ff2 can be freely controlled, thereby achieving the merging or separation of the two Q factor divergences.

[0058] Example 2:

[0059] In this embodiment, the performance of the prepared highly robust BICs biosensor is characterized based on the parameters of Example 1. First, a corresponding spatial optical path coupling system was designed and constructed, such as... Figure 4 As shown.

[0060] This system uses a supercontinuum light source. The optical signal is first transmitted through optical fiber, collimated by an optical fiber collimator, and then passes through an aperture to generate a beam with a diameter of 500 μm. The beam then passes through a beam splitter and a Glan prism to control the direction and polarization of the incident light, ultimately positioning it at the center of the sensor array. The signal reflected from the sample is transmitted again through the beam splitter, passes through a detector, and is then transmitted through optical fiber. It is collected by a spectrometer, and the reflection spectrum is detected by a photodetector. The spectrum is then displayed on a computer for processing and analysis.

[0061] Microfluidic fixture: The microfluidic channel is connected to the syringe and analyte through a Teflon tube. In actual testing, the injection speed of the syringe is controlled by a microfluidic pump to ensure that the solution in the microtube moves at a constant flow rate.

[0062] In one embodiment, the microfluidic fixture can use the structure described in patent publication number CN224037755U to fix the sensor with the HER2 recognition functional layer of this invention in the chip mounting slot of that patent. For details on the HER2 recognition functional layer on the surface of the sensor body, please refer to... Figure 9 .

[0063] Example 3:

[0064] In this embodiment, the reflectance spectrum of a highly robust BICs biosensor is obtained based on the spatial optical path coupling system built in Embodiment 2. Figure 5 The reflectance spectrum of the highly robust BICs biosensor in the resonant mode near 930-980 nm is shown, and its linewidth is subjected to Lorentz linear fitting. Its quality factor can be calculated using the following formula:

[0065] (1)

[0066] Where λ is the resonant wavelength and FWHM is the full width at half maximum (FWHM) of the resonant mode. The FWHM of the three different resonant modes of the highly robust BICs biosensor, obtained through Lorentz line fitting based on the detection spectrum, are marked in the figure. The highly robust BICs biosensor can more accurately select and enhance light at specific wavelengths, thus facilitating high-resolution spectral measurements and precise wavelength selection, enabling the detection of minute refractive index changes, and can be used for the detection of ultra-low concentrations of biomolecules.

[0067] Example 4:

[0068] To evaluate the refractive index sensing performance of the robust BICs biosensor, this case study introduced different volume ratios (0%, 2%, 4%, 6%, and 8%) of dimethyl sulfoxide (DMSO) and deionized aqueous solution into the microfluidic channel, corresponding to refractive index values ​​ranging from 1.333 to 1.342. Figure 5 The experimental reflectance spectrum and linewidth fitting spectrum of the highly robust BICs biosensor are shown. Figure 6 This study demonstrates the relationship between real-time monitoring of solution refractive index changes and resonant wavelength shift. Experimental results show that with increasing dimethyl sulfoxide (DMSO) solution concentration, the resonant wavelength exhibits a significant redshift, and a significant linear relationship exists between the resonant wavelength and the refractive index value. The sensitivity, derived from fitting the experimental data, is 160.9 nm / RIU. Furthermore, the test results maintain high robustness under long-term detection. These excellent performance indicators are attributed to the innovative design based on an all-dielectric dual-symmetry protected metasurface. During parameter tuning, it supports two different divergence points at different momentum positions. By tuning the merging of two dual-symmetry protected bioindices (BICs), it not only effectively overcomes the dependence on sporadic BICs but also utilizes the near-merging characteristics of the merged BICs to achieve a "flattened" distribution of the Q-factor within a specific parameter range. This design enables the sensor to maintain a stable optical response even in the face of fabrication errors or environmental disturbances, and to maintain stable observations under long-term complex biological processing conditions and fluid disturbances.

[0069] Example 5:

[0070] This case study uses the HER2 antigen as the target analyte, validates the assay through specific binding experiments, and performs analysis at ultra-low detection limits. The HER2 antibody-antigen protein binding assay consists of the following steps:

[0071] (1) Immerse the sample in acetone solution and heat at 70°C for 30 minutes to remove photoresist and other residues. Then, use ethanol and deionized water to clean the sample by ultrasonic cleaning two to three times to remove the acetone solution remaining on the surface. Finally, dry the sample with compressed air.

[0072] (2) Preparation of piranha solution: The volume ratio of concentrated sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) is controlled at 3:1. After H2SO4 and H2O2 are fully mixed, the solution is left to stand at room temperature.

[0073] (3) Place the dried sample into the above piranha solution and soak it at room temperature for 24 hours. This step can further remove organic residues introduced during the preparation process and at the same time generate hydroxyl groups (-OH) on the surface of the structure.

[0074] (4) Take out the sample from the piranha solution and rinse it three times with deionized water; then, sonicate it three times with deionized water for 5 minutes each time.

[0075] (5) After cleaning, the sample is dried with compressed air and then placed in a plasma cleaner for 120 seconds at a power of 67.5W. This step can further increase the hydroxyl groups (-OH) on the surface of the structure.

[0076] (6) The sample treated with oxygen plasma cleaning was placed in APTES ((3-aminopropyl)triethoxysilane) solution, the volume ratio of APTES solution to ethanol was 2%, and reacted at room temperature for 2 hours. This step allows an amino (-NH2) layer to be attached to the surface of the structure, thereby making the surface of the structure easier to capture proteins;

[0077] (7) Remove the sample and wash it with anhydrous ethanol solution, then blow it dry;

[0078] (8) Place the sample in a petri dish and spot 50 μL of 0.5 mg / mL Protein G (Recombinant) solution onto the sample surface. Let it stand in a constant humidity (50%) environment and allow it to evaporate at room temperature for 1.5 h. Then seal the petri dish and leave it overnight (20 h). After spotting, load the chip into the microfluidic channel system and use a syringe pump to draw 10 times diluted phosphate buffer solution (PBS) at a rate of 200 μL / min for 15 min to clean the unbound Protein G solution on the chip surface. Finally, use absorbent paper to dry the residual PBS solution.

[0079] (9) Place the sample in a culture dish, and spot 50 μL of HER2 antibody (recombinant anti-Her2 / ERBB2 antibody) solution at a concentration of 0.1 mg / mL on the sample surface after spotting Protein G. Let it stand in a constant humidity environment of 50% and evaporate at room temperature for 1.5 h. Then seal the culture dish and leave it overnight (20 h). After spotting, put the chip into a microfluidic fixture and use a syringe pump to draw 10 times diluted PBS at a rate of 200 μL / min for 15 min to clean the unbound HER2 antibody solution on the chip surface.

[0080] (10) Set the flow rate of the syringe pump to 30 μL / min and draw 0.1 mg / mL of BSA (Bovine Serum Albumin, BSA) solution prepared with PBS solution diluted 10 times. The purpose is to block the Protein G site of the unbound antibody by using the non-specific binding of BSA. After 15 min, keep the flow rate constant and draw PBS solution diluted 10 times to wash off the excess BSA solution. Continue until the resonance peak position remains stable. At this point, the surface functionalization modification of the chip has been completed.

[0081] (11) Then, a flow test of HER2 antigen (recombinant human Her2 / ERBB2 protein) was performed. The flow rate was maintained at 30 μL / min. 1 pM, 10 pM, 100 pM, 1 nM and 10 nM were introduced sequentially for one hour each. Finally, 10-fold diluted PBS solution was introduced for 30 min to wash away unbound HER2 antigen molecules in order to eliminate the influence of the refractive index of the antigen solution on the test results.

[0082] The design of this invention enables the sensor to maintain a stable optical response even in the face of fabrication errors or environmental disturbances. Furthermore, it maintains stable observation under prolonged and complex biological processing conditions and fluid disturbances, whereas conventional qBICs typically struggle to maintain measurability in such complex environments. This experimental phenomenon demonstrates that the design in this study improves the robustness and detection limit of this type of device. Experimental results show that the constructed sensor possesses a maximum bulk refractive index sensitivity of 160.9 nm / RIU and high robustness for long-term continuous monitoring under complex modification conditions. The robustness test graph (stability test of FWHM over time) is shown below. Figure 7 In the concentration gradient set in this embodiment, the sensor can produce a recognizable resonant wavelength response to a 1 pM HER2 antigen sample; therefore, the lowest HER2 antigen concentration that can be tested and identified is 1 pM. Figure 8 As shown. More importantly, compared to merging schemes that rely on sporadic BICs, the dual-symmetric protection mechanism adopted in this work has higher design controllability and process compatibility, providing a new path for developing label-free biosensors with high sensitivity, high resolution and high stability.

[0083] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention's specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A highly robust biosensor for in vitro HER2 antigen detection, characterized in that: The sensor body includes a substrate layer and a cell array disposed on the substrate layer. The cell array includes multiple cell unit structures. Each cell unit structure includes a convex structure formed by closely arranged first and second square pillars. The first and second square pillars have the same height. The side length of the first square pillar is w1, satisfying 180nm < w1 ≤ 250nm. The side length of the second square pillar is w2, satisfying 100nm ≤ w2 ≤ 180nm. After a HER2 recognition functional layer is disposed on the surface of the sensor body, it is integrated with a microfluidic fixture to form a highly robust biosensor for in vitro HER2 antigen detection.

2. The highly robust biosensor for in vitro HER2 antigen detection according to claim 1, characterized in that: The base layer includes a substrate and a waveguide layer; the cell array is disposed on the waveguide layer.

3. A highly robust biosensor for in vitro HER2 antigen detection according to claim 2, characterized in that: The substrate material is silicon, quartz, or glass.

4. A highly robust biosensor for in vitro HER2 antigen detection according to claim 2, characterized in that: The height of the waveguide layer is t. w Satisfying 100nm≤t w ≤250nm.

5. A highly robust biosensor for in vitro HER2 antigen detection according to claim 4, characterized in that: The t w =200nm.

6. A highly robust biosensor for in vitro HER2 antigen detection according to claim 1, characterized in that: The plurality of said unit cell structures are periodically arranged on the substrate in a first direction and a second direction, and the arrangement period of the plurality of said unit cell structures in the first direction and the second direction is P; The first direction is perpendicular to the second direction; P is 590-620nm.

7. A highly robust biosensor for in vitro HER2 antigen detection according to claim 1, characterized in that: The material of the unit cell structure is silicon nitride or titanium dioxide dielectric material; the refractive index is 1.8-2.

5.

8. A highly robust biosensor for in vitro HER2 antigen detection according to claim 1, characterized in that: The heights of the first square column and the second square column are t. g Satisfying 80nm≤t g ≤120nm.

9. A highly robust biosensor for in vitro HER2 antigen detection according to claim 8, characterized in that: The w1 = 183nm; the w2 = 122nm; the t g =100nm; the arrangement period of the unit cell structure is 610nm.

10. A highly robust biosensor for in vitro HER2 antigen detection according to claim 1, characterized in that: The HER2 recognition functional layer is formed sequentially through the following steps: hydroxylation, silanization, immobilization of Protein G, immobilization of HER2 antibody, and BSA blocking.

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