A method of detecting a biomolecule

Abstract

The application discloses a kind of detection biomolecule method, it is related to micro-nano processing technology and LSPR biosensor technical field.The existing LSPR sensor sensitivity is lower, spectral resonance peak width is larger, and the existing MIM structure processing process is complex, cost is high, modification step is complicated, and sensing performance stability is insufficient.The metal-gap-metal composite structure is constructed by the application, including preparation of a pair of sensing chips, each piece includes quartz substrate and LSPR gold nanostructure array, support structure is formed on quartz substrate, and MIM resonance cavity is formed by accurately controlling interval by buckling assembly, in combination with biological modification and detection method, active regulation of electric field intensity is realized.The application significantly improves the sensitivity of the sensor, reduces the spectral resonance peak width, simplifies the process, reduces the cost, improves the stability, and is used for the detection of trace tumor marker antigen.

Description

Technical Field

[0001] This invention belongs to the fields of micro-nano fabrication technology and LSPR biosensor technology, and particularly relates to a method for detecting biomolecules. Background Technology

[0002] Evanescent waves generated by light illuminating a noble metal surface under certain conditions couple with free electrons on the surface, leading to surface plasmon resonance (SPR). When SPR occurs, an enhanced electric field appears on the noble metal surface, and the spectral resonance peak shifts with changes in the refractive index of the medium within the electric field's coverage area. This is an excellent property with potential for label-free sensing, thus SPR-based sensors have attracted widespread research attention. Among them, refractive index sensors based on localized surface plasmon resonance (LSPR) have become an important topic in biosensing due to their ability to confine the enhanced electric field to a range of tens of nanometers on the noble metal surface and their ease of integration. However, most LSPR sensors suffer from low sensitivity and are limited by energy loss, resulting in large linewidths of their spectral resonance peaks, which significantly restricts their applications.

[0003] Currently, there are two main solutions: one is to fabricate a more complex sensor structure, such as a "metal-dielectric-metal (MIM)" structure; the other is to introduce gold-labeled modifiers in the final step of biomodification to form a "MIM"-like structure. Both improve the sensitivity of the sensor chip by enhancing the electric field and reduce light loss by forming "MIM"-like microcavities to reduce the resonance peak linewidth, thereby improving the sensing performance of LSPR biosensors. However, the former has a complex fabrication process and high cost, and it is difficult to capture a large amount of the analyte in the area with the strongest electric field (between the two metal layers) during bioprocessing, making it impossible to achieve optimal sensitivity improvement; the latter involves cumbersome modification steps, is time-consuming, and is affected by modification efficiency, making it difficult to guarantee the stability of sensing performance. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention proposes a method for detecting biomolecules. It employs semiconductor fabrication technology to prepare the sensor chip and support structure, simplifying the processing flow of traditional complex MIM structures, reducing fabrication costs, and exhibiting good process compatibility. Combining this with microfluidic chip-based biomodification ensures stable sensing performance, avoiding the cumbersome modification steps and insufficient stability issues inherent in gold labeling methods. This provides a novel technical pathway for developing high-performance LSPR biosensors.

[0005] To achieve the above objectives, the present invention provides a method for detecting biomolecules, comprising:

[0006] A pair of sensor chips are fabricated, each sensor chip comprising a quartz substrate and an array of gold nanostructures formed on the surface of the quartz substrate;

[0007] A support structure is formed on the quartz substrate of each of the sensor chips;

[0008] The surface of the gold nanostructure array is biomodified to immobilize biorecognition molecules;

[0009] The pair of sensor chips are assembled face-to-face with the gold nanostructure arrays, such that the support structure controls the spacing between the two gold nanostructure arrays, thereby forming a metal-dielectric-metal resonant cavity structure.

[0010] The assembled pair of sensor chips are fixed in the detection frame;

[0011] Measure the localized surface plasmon resonance signal of the metal-dielectric-metal resonant cavity structure;

[0012] Biomolecules are detected based on changes in the localized surface plasmon resonance signal.

[0013] Optionally, the process of fabricating a pair of sensor chips includes:

[0014] The quartz substrate was cleaned with a piranha solution and then dry-cleaned to remove surface impurities.

[0015] Photoresist is spin-coated onto a quartz substrate and pre-baked to form a photoresist layer;

[0016] Photomasks are used to expose and bake the photoresist to define the patterned areas;

[0017] The photoresist is developed using a developer to form a photoresist mask;

[0018] An adhesion layer and a gold film are grown on a photoresist mask by evaporation to form a metal layer.

[0019] Gold nanostructure arrays are formed by immersing the photoresist in acetone, peeling it off, and annealing it.

[0020] Repeated photolithography and metal deposition processes use a support structure photomask to form the support structure.

[0021] Optionally, the process of forming the supporting structure includes:

[0022] A support structure pattern is defined on a quartz substrate using photolithography, wherein the photolithographic mask pattern consists of two rectangles that are symmetrical about the central axis of the quartz substrate.

[0023] Metal is deposited on the pattern to form a support structure by a metal deposition process, wherein an adhesion layer is deposited first and then a gold film is deposited.

[0024] By precisely controlling the thickness of the metal deposition, the height of the support structure is precisely adjusted so that the height of the support structure corresponds to the interlayer spacing of the metal-dielectric-metal resonant cavity structure.

[0025] Optionally, the process of biomodifying the surface of the gold nanostructure array includes:

[0026] A microfluidic chip is provided and covered on the sensing chip to form a flow channel, wherein the microfluidic chip has multiple channels;

[0027] A solution of a thiol compound containing carboxyl groups is introduced through the flow channel to form a self-assembled monolayer via gold-sulfur bonds;

[0028] A carboxyl activator solution is introduced to activate the carboxyl groups;

[0029] A solution of a biorecognition molecule containing an amino group is introduced to fix the biorecognition molecule onto the activated carboxyl group via an amide bond;

[0030] A blocking agent solution is introduced to block unreacted active sites;

[0031] A solution of the identified biomolecule is introduced to enable the biomolecule to bind specifically to the biorecognition molecule.

[0032] Optionally, the process of assembling a pair of sensor chips together includes:

[0033] The pair of sensor chips are placed facing each other with gold nanostructure array surfaces so that the supporting structure contacts and aligns them;

[0034] The spacing between the two gold nanostructure arrays is precisely controlled by the height of the support structure, thereby forming a metal-dielectric-metal resonant cavity structure.

[0035] Ensure that the gold nanostructure array surfaces are parallel to avoid uneven electric field distribution.

[0036] Optionally, the process of fixing the assembled sensor chip to the detection frame includes:

[0037] The pair of sensor chips assembled by the snap-fit ​​are placed in the detection frame, wherein the detection frame has a structure that is open in the middle and has square ring thin sheets around the perimeter.

[0038] The pair of sensor chips are fixed by the structure of the detection frame to ensure the stability after assembly.

[0039] Optionally, the process of measuring localized surface plasmon resonance signals includes:

[0040] The sensing components, which are fixed in the detection frame, are placed inside the spectral testing system;

[0041] Measure the reflectance or transmission spectrum;

[0042] The wavelength of the local surface plasmon resonance peak is read from the spectrum.

[0043] Optionally, the process of detecting biomolecules based on signal changes includes:

[0044] Calculate the offset between the resonance peak wavelength when the biomodification is not bound to biomolecules and the resonance peak wavelength when the biomolecules are bound.

[0045] Based on the relationship between the offset and the concentration of biomolecules, qualitative and quantitative detection of the biomolecules to be tested can be achieved.

[0046] Technical advantages of this invention: This invention discloses a method for detecting biomolecules. Two sensor chips, each fabricated with an LSPR gold nanostructure array and a support structure, are assembled face-to-face within a detection frame, constructing a metal-insulator-metal (MIM) resonant cavity structure. This structure utilizes a cuboid support structure symmetrical along the central axis of a quartz substrate for assembly, simplifying the complex chip fabrication process, reducing fabrication costs, and providing strong process compatibility. The upper and lower metal layers (gold nanostructures) and the intermediate insulating dielectric layer in the MIM resonant cavity structure work together to effectively confine the electromagnetic field, reduce incident light propagation loss, and generate a strong near-field localized enhancement effect. This invention amplifies the changes in optical signals caused by the binding of biomolecules, resulting in an order-of-magnitude improvement in the sensitivity of trace tumor marker antigen detection. Simultaneously, it reduces the full width at half maximum (FWHM) of the spectral resonance peak, enhancing sensing quality. By adjusting the deposition height of the support structure, the spacing between the two gold nanostructure arrays can be precisely controlled, enabling active "customization" of the electric field strength and distribution within the sensor, achieving precise control of the electric field strength. The method and sensing chip provided by this invention offer a novel technical path for developing high-performance LSPR biosensors, combining high sensitivity, controllability, and process economy. It is suitable for precise detection scenarios of trace biomarkers, and its rational structural design facilitates efficient detection when integrated into detection systems. Attached Figure Description

[0047] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0048] Figure 1 A method for fabricating an LSPR sensor chip (15×15 sensor unit) with rectangular support strips on both sides, provided in an embodiment of the present invention;

[0049] Figure 2A cross-sectional schematic diagram of each step in the fabrication method of an LSPR sensor chip (15×15 sensor unit) with rectangular support bars on both sides, provided in an embodiment of the present invention.

[0050] Figure 3 A biomodification method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in this embodiment of the invention;

[0051] Figure 4 A cross-sectional schematic diagram of each step of the biomodification method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in an embodiment of the present invention.

[0052] Figure 5 A testing method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in an embodiment of the present invention;

[0053] Figure 6 A cross-sectional schematic diagram of each step of the testing method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in an embodiment of the present invention.

[0054] Figure 7 A schematic diagram of a photolithographic mask based on a MIM structure LSPR gold nanostructure array provided in an embodiment of the present invention;

[0055] Figure 8 A schematic diagram of a photolithographic mask for a support structure provided in an embodiment of the present invention;

[0056] Figure 9 A schematic diagram of a photolithographic mask for a microfluidic chip provided in an embodiment of the present invention;

[0057] Figure 10 A schematic diagram of a photolithographic mask for a detection framework provided in an embodiment of the present invention;

[0058] Figure 11 A three-dimensional schematic diagram of a sensor chip modified only with antibodies, provided for an embodiment of the present invention;

[0059] Figure 12 A three-dimensional schematic diagram of a sensor chip with antibodies and antigens added, provided in an embodiment of the present invention;

[0060] Figure 13 A schematic diagram of the standard curve in the test method of the tumor marker sensor based on the MIM structure LSPR sensor chip provided in the embodiment of the present invention;

[0061] Figure reference numerals: 21: Quartz substrate; 22: AR4340 photoresist; 23: Gold nanostructure array; 24: Support structure; 41: Microfluidic chip; 42: Surface-modified antibody; 43: Surface-modified antigen; 61: Detection frame. Detailed Implementation

[0062] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0063] It should be noted that the steps shown in the flowchart in the accompanying drawings can be executed in a computer system such as a set of computer-executable instructions, and although a logical order is shown in the flowchart, in some cases the steps shown or described may be executed in a different order than that shown here.

[0064] This embodiment provides a method for detecting biomolecules, including:

[0065] A pair of sensor chips are fabricated, each sensor chip comprising a quartz substrate and an array of gold nanostructures formed on the surface of the quartz substrate;

[0066] A support structure is formed on the quartz substrate of each of the sensor chips;

[0067] The surface of the gold nanostructure array is biomodified to immobilize biorecognition molecules;

[0068] The pair of sensor chips are assembled face-to-face with the gold nanostructure arrays, such that the support structure controls the spacing between the two gold nanostructure arrays, thereby forming a metal-dielectric-metal resonant cavity structure.

[0069] The assembled pair of sensor chips are fixed in the detection frame;

[0070] Measure the localized surface plasmon resonance signal of the metal-dielectric-metal resonant cavity structure;

[0071] Biomolecules are detected based on changes in the localized surface plasmon resonance signal.

[0072] Furthermore, the process of fabricating a pair of sensor chips includes:

[0073] The quartz substrate was cleaned with a piranha solution and then dry-cleaned to remove surface impurities.

[0074] Photoresist is spin-coated onto a quartz substrate and pre-baked to form a photoresist layer;

[0075] Photomasks are used to expose and bake the photoresist to define the patterned areas;

[0076] The photoresist is developed using a developer to form a photoresist mask;

[0077] An adhesion layer and a gold film are grown on a photoresist mask by evaporation to form a metal layer.

[0078] Gold nanostructure arrays are formed by immersing the photoresist in acetone, peeling it off, and annealing it.

[0079] Repeated photolithography and metal deposition processes use a support structure photomask to form the support structure.

[0080] Furthermore, the process of forming the supporting structure includes:

[0081] A support structure pattern is defined on a quartz substrate using photolithography, wherein the photolithographic mask pattern consists of two rectangles that are symmetrical about the central axis of the quartz substrate.

[0082] Metal is deposited on the pattern to form a support structure by a metal deposition process, wherein an adhesion layer is deposited first and then a gold film is deposited.

[0083] By precisely controlling the thickness of the metal deposition, the height of the support structure is precisely adjusted so that the height of the support structure corresponds to the interlayer spacing of the metal-dielectric-metal resonant cavity structure.

[0084] Furthermore, the process of biomodifying the surface of gold nanostructure arrays includes:

[0085] A microfluidic chip is provided and covered on the sensing chip to form a flow channel, wherein the microfluidic chip has multiple channels;

[0086] A solution of a thiol compound containing carboxyl groups is introduced through the flow channel to form a self-assembled monolayer via gold-sulfur bonds;

[0087] A carboxyl activator solution is introduced to activate the carboxyl groups;

[0088] A solution of a biorecognition molecule containing an amino group is introduced to fix the biorecognition molecule onto the activated carboxyl group via an amide bond;

[0089] A blocking agent solution is introduced to block unreacted active sites;

[0090] A solution of the identified biomolecule is introduced to enable the biomolecule to bind specifically to the biorecognition molecule.

[0091] Furthermore, the process of assembling a pair of sensor chips together includes:

[0092] The pair of sensor chips are placed facing each other with gold nanostructure array surfaces so that the supporting structure contacts and aligns them;

[0093] The spacing between the two gold nanostructure arrays is precisely controlled by the height of the support structure, thereby forming a metal-dielectric-metal resonant cavity structure.

[0094] Ensure that the gold nanostructure array surfaces are parallel to avoid uneven electric field distribution.

[0095] Furthermore, the process of fixing the assembled sensor chip to the detection frame includes:

[0096] The pair of sensor chips assembled by the snap-fit ​​are placed in the detection frame, wherein the detection frame has a structure that is open in the middle and has square ring thin sheets around the perimeter.

[0097] The pair of sensor chips are fixed by the structure of the detection frame to ensure the stability after assembly.

[0098] Furthermore, the process of measuring localized surface plasmon resonance signals includes:

[0099] The sensing components, which are fixed in the detection frame, are placed inside the spectral testing system;

[0100] Measure the reflectance or transmission spectrum;

[0101] The wavelength of the local surface plasmon resonance peak is read from the spectrum.

[0102] Furthermore, the process of detecting biomolecules based on signal changes includes:

[0103] Calculate the offset between the resonance peak wavelength when the biomodification is not bound to biomolecules and the resonance peak wavelength when the biomolecules are bound.

[0104] Based on the relationship between the offset and the concentration of biomolecules, qualitative and quantitative detection of the biomolecules to be tested can be achieved.

[0105] Specifically, the implementation process of this embodiment includes:

[0106] The present invention provides a highly sensitive local surface plasmon resonance (LSPR) sensing chip, comprising: a pair of sensing chips disposed opposite to each other, each sensing chip comprising a quartz substrate and an array of LSPR gold nanostructures formed on the surface of the quartz substrate;

[0107] A pair of support structures are formed on both sides of the quartz substrate of each of the sensor chips. The height of the support structures is fabricated by semiconductor technology. When a pair of sensor chips are assembled with gold nanostructure arrays interlocked, the spacing between the gold nanostructure arrays can be precisely controlled, thereby forming a metal-insulator-metal (MIM) resonant cavity structure.

[0108] A detection frame is used to house and secure the pair of sensor chips assembled together.

[0109] According to the MIM structure LSPR sensing chip provided by the present invention, the sensing chip consists of 15×15 sensing units arranged in a matrix, and each sensing unit is composed of an array of gold nanostructures.

[0110] According to the MIM structure LSPR sensor chip provided by the present invention, the support structure is a cuboid support strip symmetrically arranged along the central axis of the quartz substrate, which is used to adjust the spacing between a pair of LSPR sensor chips.

[0111] According to the MIM structure LSPR sensing chip provided by the present invention, the precise spacing is achieved by adjusting the deposition height of the support structure, the range of which enables the MIM resonant cavity structure to generate a local electric field enhancement effect.

[0112] The method for fabricating a MIM structure LSPR sensing chip according to the present invention includes the following steps: an LSPR gold nanostructure array can be formed on a quartz substrate by semiconductor process technology;

[0113] The supporting structure is formed on the same quartz substrate on which the gold nanostructure array is formed, thereby obtaining the sensing chip.

[0114] According to the preparation method provided by the present invention, the metal nanostructure preparation method adopts self-absorption nanoimprinting, nanoimprinting and rapid annealing to form a metal nanostructure array, and deposits an adhesion layer before depositing a gold film.

[0115] In specific embodiments, the figures are explained as follows:

[0116] Figure 1 A method for fabricating an LSPR sensor chip (15×15 sensor unit) with rectangular support strips on both sides, provided in an embodiment of the present invention;

[0117] Figure 2 The following are cross-sectional schematic diagrams corresponding to each step of the fabrication method of the LSPR sensor chip (15×15 sensor unit) with rectangular support strips on both sides provided in the embodiments of the present invention, as shown in the figure. Figure 2 As shown, the structure sequentially displays a quartz substrate 21 (S101), a substrate after spin-coating AR4340 photoresist 22 (S102), a 15×15 sensing unit region (S103), a discrete gold nanostructure after depositing metal (2nm Cr, 10nm Au) (S104), a structure forming a gold nanostructure array 23 after photoresist removal (S105), and a complete sensing chip after fabricating a support structure 24 (S106) (including processes such as photoresist coating, photolithography, metal evaporation, and lift-off).

[0118] Figure 3A biomodification method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in this embodiment of the invention;

[0119] Figure 4 This is a cross-sectional schematic diagram of each step in the biomodification method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in an embodiment of the present invention, as shown below. Figure 4 As shown, the structure includes a quartz substrate 21, a gold nanostructure array 23, a microfluidic chip 41 (S201-S202) covering the sensing chip, and surface-modified antibodies 42 and antigens 43 formed sequentially through a modification process, which are respectively a surface functionalized modification layer (S203), a surface-modified antibody layer (S204-S205), a blocked modification layer (S206), and a surface-modified antigen layer (S207).

[0120] Figure 5 A testing method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in an embodiment of the present invention;

[0121] Figure 6 The following are cross-sectional schematic diagrams corresponding to each step of the testing method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in this embodiment of the invention, as shown in the figure. Figure 6 As shown, a pair of modified sensor chips are assembled with the LSPR gold nanostructure array surfaces facing each other and then fixed in the detection frame 61 to form a metal-insulator-metal (MIM) resonant cavity structure. This ensures that the gold nanostructure array surfaces are parallel to avoid uneven electric field distribution and is used for the acquisition of reflection or transmission spectral signals.

[0122] Figure 7 This is a schematic diagram of a photolithographic mask based on a MIM-structured LSPR gold nanostructure array provided in an embodiment of the present invention, as shown below. Figure 7 As shown, the mask pattern is a square that can just cover the entire quartz substrate, corresponding to a 15×15 sensing unit area, which is used to fabricate an LSPR gold nanostructure array on the surface of the sensing chip.

[0123] Figure 8 A schematic diagram of the photolithographic mask for the support structure provided in an embodiment of the present invention, as shown below. Figure 8 As shown, the mask pattern consists of two rectangles that are axially symmetrical along the central axis of the quartz substrate. These rectangles are used to fabricate cuboid support bars. By adjusting the metal deposition height, a chip spacing of 10-50 nm can be achieved to generate a local electric field enhancement effect.

[0124] Figure 9 This is a schematic diagram of the photolithographic mask for a microfluidic chip provided in an embodiment of the present invention, as shown below. Figure 9As shown, the mask pattern contains a 15-channel design, corresponding to the flow channels of the microfluidic chip with a height of 50-100μm and a width of 100-300μm, as well as sample loading / effervescence orifices with a diameter of 400-600μm, to ensure uniform solution distribution and reduce dead volume;

[0125] Figure 10 A schematic diagram of the photolithographic mask for the detection framework provided in an embodiment of the present invention, as shown below. Figure 10 As shown, the mask pattern is a square with a central through-hole and a square ring thin sheet around the perimeter. It is used to prepare a detection frame 61 that can accommodate and fix the snap-fit ​​sensor chip, ensuring the stability of the chip after assembly.

[0126] Figure 11 This is a three-dimensional schematic diagram of a sensor chip modified only with antibodies, provided in an embodiment of the present invention, as shown below. Figure 11 As shown, the sensing chip includes a quartz substrate 21, a gold nanostructure array 23 formed on the surface of the quartz substrate 21, a cuboid support strip 24 symmetrical along the central axis of the substrate, and a surface modifier 42 containing only antibodies fixed on the surface of the gold nanostructure array 23 by gold-sulfur bonds and amide bonds.

[0127] Figure 12 A three-dimensional schematic diagram of a sensor chip with antibodies and antigens added, provided in an embodiment of the present invention, is shown below. Figure 12 As shown, the sensor chip structure is identical to that of the chip with only antibody added (containing a quartz substrate 21, a gold nanostructure array 23, and a support structure 24). Its surface modification 43 is a complex formed by the specific binding of the antibody and the antigen, used to react with the blank chip ( Figure 11 The spectral data of the two samples were compared to calculate the resonance peak shift.

[0128] Figure 13 This is a schematic diagram of the standard curve in the testing method of the tumor marker sensor based on the MIM structure LSPR sensor chip provided in the embodiments of the present invention, as shown in the figure. Figure 13 As shown in the image, The LSPR resonance peak wavelength is the wavelength when the sensor chip is modified only when the antibody is not bound to the antigen. The wavelength of the LSPR resonance peak after the sensor chip is incubated with the test solution and the antibody binds to the antigen is the value of the difference between the two resonance peaks. , The size is directly proportional to the concentration of the tumor marker antigen solution.

[0129] In this embodiment of the invention, the core structure of the sensing chip is an LSPR gold nanostructure array on a quartz substrate, which is fabricated using semiconductor technology.

[0130] Optionally, the LSPR gold nanostructure array consists of 15×15 sensing units arranged in a matrix, with the corresponding photomask pattern being a square that can just cover the entire quartz substrate. Figure 7 As shown, Figure 7 A schematic diagram of a photolithographic mask for an LSPR gold nanostructure array provided in an embodiment of the present invention.

[0131] Optionally, the support structure is a cuboid support strip symmetrically arranged along the central axis of the quartz substrate, and its photomask pattern is two rectangles symmetrically arranged along the central axis of the chip, such as... Figure 8 As shown, this is used to precisely control the spacing between a pair of sensor chips.

[0132] Optionally, the length and width of the quartz substrate of the reaction layer can be adjusted according to the detection requirements. A transparent hard quartz material of 20mm×20mm is usually selected to ensure the light transmittance of optical detection.

[0133] Optionally, during the metal deposition process, an adhesion layer is deposited first, followed by a gold film. The adhesion layer can be made of 5nm thick chromium, and the gold film is 100nm thick. The specific thickness depends on the chip size and the thickness of the surface modification layer. The adhesion layer and the gold film can also be replaced with other suitable materials according to actual needs.

[0134] Optionally, the height of the support structure can be achieved by adjusting the metal deposition thickness, so that the spacing between the two assembled sensor chips can generate a local electric field enhancement effect. The spacing range corresponding to the conventional deposition height is 10-50nm.

[0135] Example 1

[0136] Based on the above embodiments, the present invention also provides a method for fabricating a LSPR sensor chip based on a MIM structure, specifically including the following steps:

[0137] Step S101: After cleaning with a standard piranha solution, dry cleaning is performed: the quartz substrate is cleaned and placed in an oxygen plasma degumming machine for surface treatment. The system is programmed to use nitrogen 300 mL / min, oxygen 300 mL / min, power 300W, and time 2 minutes to remove surface impurities and activate the surface.

[0138] Step S102: Evaporate HMDS to improve photoresist adhesion and avoid photoresist drift; Place the chip in a spin coater and spin coat AR4340 photoresist. Spin coat conditions are: initial rotation speed 1000 rpm for 6 seconds, high-speed rotation speed 6000 rpm for 20 seconds; then place it on a hot plate for pre-baking at 110°C for 2 minutes.

[0139] Step S103: Expose the photoresist using the designed photomask (15×15 sensing unit area) at an exposure power of 8.2mw / cm², then place it on a hot plate for post-baking at 100℃ for 80 seconds; develop it with a KMP PD238 developer solution with a water ratio of 5:2 for 90 seconds to obtain the photoresist mask.

[0140] Step S104: An adhesion layer and a gold film are grown on the above product using an evaporation method. The adhesion layer has a thickness of 5 nm, and the gold film has a thickness of 100 nm.

[0141] Step S105: Immerse the product in acetone to peel it off, and then use ultrasound, degumming, and annealing to form a gold nanostructure array;

[0142] Step S106: Repeat steps S102-S105, using a photomask containing two symmetrical rectangular patterns to prepare a support structure, thus completing the fabrication of the sensor chip.

[0143] In the above preparation process, in addition to thermal evaporation combined with photolithography lift-off, metal nanostructures can also be prepared by self-absorption nanoimprinting, nanoimprinting, rapid annealing and other methods to form metal nanostructure arrays. The specific process can be selected according to the laboratory equipment and precision requirements.

[0144] Example 2

[0145] Based on the aforementioned MIM structure LSPR sensing chip, this invention also provides a corresponding surface modification method, which uses a microfluidic chip to assist in the immobilization of biorecognition molecules. The specific process is detailed in the appendix. Figure 3 , 4 9. Explanation, among which Figure 3 The present invention provides a biomodification method for a tumor marker sensor based on a MIM structure LSPR sensor chip. Figure 4 This is a cross-sectional schematic diagram of each step in the biomodification method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in an embodiment of the present invention. Figure 9 A schematic diagram of a photolithographic mask for fabricating a microfluidic chip provided in an embodiment of the present invention includes the following steps:

[0146] Step S201, Microfluidic chip fabrication: Clean the quartz substrate, spin-coat photoresist and dry it; expose and develop the designed photomask (containing a pattern of 15 channels) to obtain the photoresist mask; form a three-dimensional microfluidic chip substrate by wet etching; peel it off by acetone soaking to form the main structure of the microfluidic chip; soak and clean it with a mixed solution of hydrogen peroxide and concentrated sulfuric acid with a volume ratio of 1:3, rinse it repeatedly with deionized water, blow it dry with nitrogen, and then treat it with oxygen plasma (parameters are the same as those for chip substrate treatment).

[0147] Step S202: Assemble the modification chip: The microfluidic chip is horizontally covered on the sensor chip to assemble it into a modification chip.

[0148] Step S203, Monolayer Formation: Dissolve 11-mercaptoundecanoic acid (11-MUA) in anhydrous ethanol to prepare a 15 mM solution. Immerse the modified chip in the above solution and react at 27°C for 2 hours to fix the carboxyl groups on the surface of the sensor chip using gold-sulfur bonds. After the reaction is complete, wash the combined chip with anhydrous ethanol and phosphate buffer (PBS).

[0149] Step S204, Carboxyl activation: Dissolve 400mM EDC and 100mM NHS separately in MES solution, mix them in equal volumes, and immerse the combined chip in the solution for 30 minutes to activate the carboxyl groups; then wash with PBS buffer.

[0150] Step S205, Antibody fixation: Add an amino-containing tumor marker antibody (such as alpha-fetoprotein AFP antibody), and react in a shaker at 27°C for 2 hours to allow the amino group to bind to the activated carboxyl group through an amide bond, thus fixing the antibody onto the chip surface; wash with PBS buffer.

[0151] Step S206: Block unreacted sites: Add 10% ethanolamine solution and react in a shaker at 27°C for 1 hour to block unbound active sites; wash with PBS buffer.

[0152] Step S207, Antigen Binding: After removing the microfluidic chip, longitudinally cover it on the sensing chip, add the corresponding tumor marker antigen solution, and react in a shaker at 27°C for 1 hour to allow the antigen and antibody to bind specifically; wash with PBS buffer.

[0153] Optionally, the microfluidic chip has a microchannel height of 50-100 μm, a width of 100-300 μm, and a sample loading orifice and an effluent orifice diameter of 400-600 μm to ensure uniform solution distribution and reduce dead volume.

[0154] Optionally, the type of antibody is selected based on the tumor marker being detected, such as using AFP antibody to detect AFP, or CEA antibody to detect carcinoembryonic antigen (CEA), and the antigen must have a specific binding relationship with the antibody.

[0155] Optionally, the temperature of each reaction step can be controlled at room temperature (25-30℃), and the reaction time can be adjusted appropriately according to the antibody / antigen affinity. The conventional antibody fixation time is 1-2 hours, and the antigen binding time is 0.5-1 hour.

[0156] Example 3

[0157] After the above modifications are completed, the present invention provides a corresponding LSPR sensing detection method, combined with the appendix. Figure 5 ,6 Explanation of points 10 and 13, among which... Figure 5 The present invention provides a testing method for a tumor marker sensor based on a MIM structure LSPR sensor chip. Figure 6 This is a cross-sectional schematic diagram of each step in the testing method for a tumor marker sensor based on a MIM structure LSPR sensor chip provided in an embodiment of the present invention. Figure 10 This is a schematic diagram of the photolithographic mask for the detection framework provided in an embodiment of the present invention. Figure 13 This is a schematic diagram of the test curve in the tumor marker testing method based on a MIM structure LSPR sensor chip provided in an embodiment of the present invention. The specific steps are as follows:

[0158] Step S301: Dry the two sensor chips modified with antibodies or antigens with a nitrogen gun, assemble the support structure with the chips facing each other, install them in the detection frame, and fix them in the test frame.

[0159] Step S302: First, test the dark spectrum and reference spectrum, then perform signal spectrum testing. Place the assembled sensor chip (modified with antibody) into the LSPR spectral testing system, measure the reflectance spectrum or transmission spectrum, and read the resonance peak wavelength. .

[0160] Step S303: Connect the two antigen-modified sensor chips together and assemble and fix them as described above, then measure the resonance peak wavelength of the spectrum. .

[0161] Step S304: Calculate the resonance peak shift. Analyze the changes in concentration and wavelength of the target analyte. The relationship between them.

[0162] Preferably, during the detection process, the chip assembly must ensure that the gold nanostructure array surfaces are opposite and parallel to each other to avoid uneven electric field distribution due to tilting, which would affect the detection accuracy.

[0163] Optionally, LSPR spectroscopy testing can be performed using either reflectance or transmission spectroscopy modes, depending on the chip structure and light source characteristics. Reflectance spectroscopy mode is typically used to facilitate rapid reading of peak position shifts.

[0164] Optionally, the standard curve is prepared by using antigen solutions of different concentrations, and the results are detected and recorded according to the method described above. Plotting antigen concentration on the x-axis, Plot the vertical axis and use A standard curve equation is obtained by performing linear fitting, which is used to quantitatively calculate the concentration of the sample to be tested.

[0165] Based on any of the above embodiments, the present invention also provides specific application embodiments: taking the detection of alpha-fetoprotein (AFP) as an example, the quartz substrate is selected with a size of 20mm×20mm×1.5mm, the gold nanostructure array is a 15×15 matrix, the support structure is a chromium / gold cuboid strip, and the microfluidic chip contains 15 channels; during the modification process, the 11-MUA concentration is 15mM, the reaction time is 4 hours (constant temperature and humidity), the EDC / NHS activation time is 40 minutes, the AFP antibody reaction time is 2 hours, the ethanolamine blocking time is 1 hour, and the AFP antigen reaction time is 40 minutes; during detection, the resonance peak wavelengths of the blank and the sample are measured respectively, the offset is calculated, and the AFP concentration is obtained by comparing with the standard curve.

[0166] This invention provides a MIM-based LSPR sensor chip and detection method. Two sensor chips are assembled by interlocking using a symmetrical cuboid support structure to construct a MIM resonant cavity structure. This simplifies the fabrication process of traditional MIM structures, reduces manufacturing costs, and offers strong process compatibility. The MIM structure effectively confines the electromagnetic field, reduces incident light propagation loss, and generates a strong near-field local enhancement effect, resulting in an order-of-magnitude improvement in the detection sensitivity of trace tumor biomarkers, while simultaneously reducing the resonance peak's full width at half maximum (FWHM). The spacing can be precisely controlled by adjusting the height of the support structure, enabling active customization of the electric field strength and distribution. This chip, combined with microfluidic modification technology, requires a small sample volume per detection and achieves precise quantification through spectral peak shifting. Its simple structure and ease of integration make it suitable for rapid on-site detection of trace biomarkers.

[0167] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for detecting biomolecules, characterized in that, include: A pair of sensor chips are fabricated, each sensor chip comprising a quartz substrate and an array of gold nanostructures formed on the surface of the quartz substrate; A support structure is formed on the quartz substrate of each of the sensor chips, the support structure being two cuboid metal support strips symmetrically arranged along the central axis of the quartz substrate; The process of forming the support structure includes: defining a support structure pattern on a quartz substrate using photolithography, wherein the photolithographic mask pattern consists of two rectangles symmetrical about the central axis of the quartz substrate; depositing metal on the pattern using metal deposition to form the support structure, wherein an adhesion layer is deposited first and then a gold film is deposited; and precisely controlling the metal deposition thickness to precisely adjust the height of the support structure so that the height of the support structure corresponds to the interlayer spacing of the metal-dielectric-metal resonant cavity structure, wherein the interlayer spacing is 10-50 nm. The surface of the gold nanostructure array is biomodified to immobilize biorecognition molecules; The pair of sensor chips are assembled by interlocking them with their gold nanostructure array surfaces facing each other, such that the support structure controls the spacing between the two gold nanostructure arrays, thereby forming a metal-dielectric-metal resonant cavity structure. The process of interlocking the pair of sensor chips includes: placing the pair of sensor chips with their gold nanostructure array surfaces facing each other, so that the support structure contacts and aligns them; precisely controlling the spacing between the two gold nanostructure arrays by the height of the support structure, thereby forming a metal-dielectric-metal resonant cavity structure; and ensuring that the gold nanostructure array surfaces are parallel to avoid uneven electric field distribution. The assembled pair of sensor chips are fixed in the detection frame; the process of fixing the assembled sensor chips in the detection frame includes: placing the assembled pair of sensor chips in the detection frame, wherein the detection frame has a structure with a through center and square rings around the perimeter; the pair of sensor chips are fixed by the structure of the detection frame to ensure the stability after assembly; Measure the localized surface plasmon resonance signal of the metal-dielectric-metal resonant cavity structure; Biomolecules are detected based on changes in the localized surface plasmon resonance signal.

2. The method for detecting biomolecules as described in claim 1, characterized in that, The process of fabricating a pair of sensor chips includes: The quartz substrate was cleaned with a piranha solution and then dry-cleaned to remove surface impurities. Photoresist is spin-coated onto a quartz substrate and pre-baked to form a photoresist layer; Photomasks are used to expose and bake the photoresist to define the patterned areas; The photoresist is developed using a developer to form a photoresist mask; An adhesion layer and a gold film are grown on a photoresist mask by evaporation to form a metal layer. Gold nanostructure arrays are formed by immersing the photoresist in acetone, peeling it off, and annealing it. Repeated photolithography and metal deposition processes use a support structure photomask to form the support structure.

3. The method for detecting biomolecules as described in claim 1, characterized in that, The process of biomodifying the surface of gold nanostructure arrays includes: A microfluidic chip is provided and covered on the sensing chip to form a flow channel, wherein the microfluidic chip has multiple channels; A solution of a thiol compound containing carboxyl groups is introduced through the flow channel to form a self-assembled monolayer via gold-sulfur bonds; A carboxyl activator solution is introduced to activate the carboxyl groups; A solution of a biorecognition molecule containing an amino group is introduced to fix the biorecognition molecule onto the activated carboxyl group via an amide bond; A blocking agent solution is introduced to block unreacted active sites; A solution of the identified biomolecule is introduced to enable the biomolecule to bind specifically to the biorecognition molecule.

4. The method for detecting biomolecules as described in claim 1, characterized in that, The process of measuring localized surface plasmon resonance signals includes: The sensing components, which are fixed in the detection frame, are placed inside the spectral testing system; Measure the reflectance or transmission spectrum; The wavelength of the local surface plasmon resonance peak is read from the spectrum.

5. The method for detecting biomolecules as described in claim 1, characterized in that, The process of detecting biomolecules based on signal changes includes: Calculate the offset between the resonance peak wavelength when the biomodification is not bound to biomolecules and the resonance peak wavelength when the biomolecules are bound. Based on the relationship between the offset and the concentration of biomolecules, qualitative and quantitative detection of the biomolecules to be tested can be achieved.

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