High-sensitivity optical biochip based on AAO and ZIF-8 composite structure and preparation and detection method thereof

Abstract

The invention discloses a high-sensitivity optical biochip based on an AAO and ZIF-8 composite structure and a preparation and detection method thereof, and relates to the technical field of optical biochip detection. The preparation method comprises the following steps: coating the surface of nanopore aluminum oxide with ZIF-8 to obtain a ZIF-8 layer on the surface of the nanopore aluminum oxide, then carrying out carboxyl or amino modification to obtain a functionalized ZIF-8 layer, and then fixing a specific antibody on the surface of the functionalized ZIF-8 layer by using a cross-linking agent to obtain the optical biosensor. According to the sensor, an AAO nanopore array serves as a structural substrate, a high-activity MOF functional layer is integrated, ultra-sensitive detection is achieved through the precise pore channel confinement effect and the molecular recognition technology, and the prepared optical biosensor has the advantages of being low in cost, high in stability, short in detection time, high in detection sensitivity and specificity and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of optical biochip detection, and in particular to a highly sensitive optical biochip based on a composite structure of AAO and ZIF-8, and a preparation and detection method thereof. Background Art

[0002] Cd-DTPA is a stable chelate formed by cadmium ions (Cd 2+ ) and diethylenetriaminepentaacetic acid (DTPA), which is a common metal-organic complex and widely exists in industrial wastewater and mine drainage. As an efficient chelating agent, DTPA can form a stable multi-dentate coordination structure with cadmium ions, thus significantly increasing the solubility and mobility of cadmium. This property makes Cd-DTPA play an important role in the environment: on the one hand, its formation may reduce the direct toxicity of free Cd 2+ ; on the other hand, it enhances the migration ability of cadmium, enabling it to diffuse over long distances and penetrate into the groundwater system, forming secondary pollution and significantly increasing the difficulty of environmental remediation.

[0003] The harm of Cd-DTPA in the environment is mainly reflected in its long-term stability and potential toxicity. Although chelated cadmium shows relatively low biotoxicity in some cases, in acidic or strongly oxidizing environments, Cd-DTPA may dissociate, releasing highly toxic free Cd 2+ , posing a serious threat to water and soil ecosystems. In addition, Cd-DTPA can contaminate groundwater through infiltration or runoff, affecting drinking water safety, and enter the food chain through bioaccumulation, causing long-term harm to the health of humans and animals. Cadmium is a known carcinogen, and its excessive exposure can lead to kidney damage, bone lesions and other health problems. The stable existence of Cd-DTPA makes it difficult for traditional pollution control technologies (such as precipitation or adsorption methods) to effectively remove cadmium, further exacerbating the difficulty of its treatment.

[0004] Currently, the technical methods for detecting Cd-DTPA are diverse, mainly including spectroscopic analysis, electrochemical detection, and chromatographic techniques, etc. Spectroscopic methods (such as ultraviolet-visible absorption spectroscopy and atomic absorption spectroscopy) identify Cd-DTPA by measuring the absorbance or luminescence intensity at specific wavelengths; electrochemical methods (such as anodic stripping voltammetry) achieve detection by measuring the change in current signals; liquid chromatography (such as high-performance liquid chromatography, HPLC) combined with mass spectrometry can achieve highly sensitive analysis of Cd-DTPA. In addition, nanotechnologies developed in recent years, such as nanopore sensors and reflectometric interference spectroscopy (RIfS), have also become potential tools for detecting Cd-DTPA due to their rapid response and high sensitivity. The detection ranges of different methods are usually between μg / L and ng / L, and the emerging nanotechnologies can achieve lower detection limits, which are suitable for trace pollutant monitoring.

[0005] The monitoring and treatment of Cd-DTPA are of great significance for environmental protection and pollution remediation. It can not only evaluate the pollution scope and severity but also provide a scientific basis for formulating effective treatment strategies. Summary of the Invention

[0006] The purpose of the present invention is to provide a highly sensitive optical biochip based on the composite structure of nanoporous alumina AAO and ZIF-8, as well as its preparation and detection methods, to solve the problems existing in the above-mentioned prior art. This optical biochip has the advantages of low cost, high stability, short detection time, high detection sensitivity and specificity, etc., providing technical support for the rapid detection of complex heavy metal complexes.

[0007] The present invention combines the metal-organic framework material (ZIF-8) with regular nanoporous anodic alumina (AAO) to construct an efficient sensing interface, prepares an optical biochip for detecting Cd-DTPA, and uses reflectometric interference spectroscopy (RIfS) to achieve the detection of Cd-DTPA. This optical biochip not only makes full use of the structural regularity of the AAO membrane and the high specific surface area of ZIF-8 but also improves the immobilization efficiency of antibodies and the capture ability of target substances through surface functionalization, providing technical support for the rapid detection of complex heavy metal complexes.

[0008] The present invention provides the following solutions:

[0009] One of the technical solutions of the present invention: Provide a preparation method of an optical biochip, including the following steps:

[0010] (1) Coat ZIF-8 on the surface of nanoporous alumina to form a ZIF-8 layer on the surface of nanoporous alumina;

[0011] (2) Modify the surface of the ZIF-8 layer with carboxyl or amine groups to obtain a functionalized ZIF-8 layer;

[0012] (3) Fix the specific antibody on the surface of the functionalized ZIF-8 layer using a crosslinking agent to obtain the optical biochip.

[0013] Furthermore, the nanoporous alumina has a regular pore array structure.

[0014] Furthermore, the coating method is preferably spin coating.

[0015] The pore size of the nanoporous alumina with a regular pore array structure used in the present invention is preferably between 150 - 500 nm, and the pore depth is preferably 15 - 20 μm. Further preferably, it is a hexagonal array with a pore spacing of 500 - 1000 nm.

[0016] The preparation method of the nanoporous alumina includes the following steps:

[0017] Design a regular pore array pattern on the surface of the aluminum substrate and perform chemical etching to obtain an aluminum substrate template with a regular pore array, and then perform anodic oxidation to obtain the nanoporous alumina.

[0018] Furthermore, the solution used for anodic oxidation is an oxalic acid solution.

[0019] Furthermore, the concentration of the oxalic acid solution is 0.3 M, the voltage is 40 - 60 V, the temperature is 5 - 10 °C, and the time is 1 - 2 h.

[0020] More specifically, the preparation process of the nanoporous alumina is as follows: First, select high-purity aluminum (purity ≥ 99.99%) as the substrate material and perform surface pretreatment to make the surface smooth through mechanical polishing or electrochemical polishing. Electrochemical polishing usually uses an acid solution to treat at a voltage of 20 V for 2 - 5 minutes to obtain a mirror effect. Subsequently, uniformly coat a layer of positive photoresist with a thickness of 1 - 2 μm on the aluminum surface, and use a photolithography machine to expose it with ultraviolet light with a wavelength of 405 nm or 365 nm. The exposure energy is 50 - 100 mJ / cm 2 , and generate a photolithography pattern of a regular pore array according to the design. After development, selectively chemically etch the exposed area of the aluminum with a 1 - 3 wt% phosphoric acid solution or chloride solution for 1 - 3 minutes to form a regular initial template. Subsequently, place the templated aluminum substrate in an oxalic acid solution (0.3 M) for anodic oxidation, with a voltage of 40 - 60 V, the temperature controlled at 5 - 10 °C, and the time of 1 - 2 hours, so that the pore array preferentially expands along the position of the photolithography template to form regular nanoporous AAO.

[0021] The preparation process of the more specific functional layer is as follows: Prepare the ZIF-8 precursor solution by dissolving 2-methylimidazole and zinc nitrate hexahydrate in methanol respectively, with a concentration usually ranging from 0.1M to 0.2M, and stir well to make them evenly mixed. Subsequently, place the prepared AAO on a spin-coating device and spin-coat the ZIF-8 precursor solution at a speed of 2000 - 3000 rpm for 30 - 60 seconds to evenly cover the surface of AAO with the solution. After spin-coating, let the sample stand at room temperature for drying, or dry it in an oven at 50 - 80°C for 10 - 30 minutes to promote the uniform growth of ZIF-8 crystals on the surface of AAO. After drying, a dense and evenly covered ZIF-8 thin film is formed, providing an ideal functional layer for subsequent biofunctionalization or detection applications.

[0022] Further, in step (2), carboxyl modification is carried out using a carboxyl modifier, and the carboxyl modifier is terephthalic acid (TPA), citric acid, or oxalic acid.

[0023] The carboxyl modifier used in the present invention has multiple carboxyl groups and can undergo a coordination reaction with metal nodes (such as Zn2+) on the surface of ZIF-8.

[0024] Further, dissolve the modifier in a suitable solvent, such as ethanol, dimethylformamide (DMF), or deionized water, to form a uniform reaction solution. Subsequently, immerse the ZIF-8 material in the modifier solution and incubate it at room temperature or under appropriately elevated temperature conditions (such as 50 - 80°C) for a period of time (preferably 30 min) to promote the binding of the carboxyl modifier to the surface of ZIF-8.

[0025] In step (2), amino modification is carried out using an amino modifier; the amino modifier can be selected from ethylenediamine or 1,6-hexanediamine.

[0026] Further, the specific antibody is a Cd-DTPA specific antibody.

[0027] The second technical solution of the present invention: Provide an optical biochip prepared by the above preparation method.

[0028] Compared with ZIF-8, other types of porous framework structure materials (such as MIL-53, HKUST-1, etc.) show obvious disadvantages in the application of the present invention. The specific reasons are as follows:

[0029] 1) Insufficient pore size and pore volume

[0030] The pore size of ZIF-8 is about It can effectively capture and immobilize target molecules. Compared with other porous materials, the pore size and pore volume characteristics of ZIF-8 are more suitable for the highly sensitive biochip developed in the present invention. For example, HKUST-1 has a relatively large pore size (about ), resulting in insufficient selectivity and capture ability for small molecules and being unable to effectively amplify the signal change of reflection interference spectroscopy.

[0031] 2) Lower thermal and chemical stability

[0032] Many other porous materials are prone to degradation or loss of structural integrity in humid or complex biological environments. For example, MIL-53 has poor stability in aqueous solutions and is prone to structural collapse, resulting in a significant decline in the signal enhancement effect of the chip. However, the ZIF-8 material used in the present invention exhibits excellent thermal and chemical stability under various environmental conditions, ensuring the long-term reliability of the chip.

[0033] 3) Convenience of functionalization

[0034] ZIF-8 has abundant coordination nodes (such as Zn2+) and active functional groups (such as imidazole groups). Functional groups such as carboxyl groups and amine groups can be easily introduced through surface modification, facilitating the immobilization of biomolecules such as antibodies and enzymes. In the process of functionalization of other porous materials, more complex chemical modification steps are often required, and the immobilization efficiency and stability of the modified materials for biomolecules are inferior to those of the ZIF-8 material used in the present invention.

[0035] The third technical solution of the present invention: Provide the application of the above optical biochip in the detection of metal-complexes, environmental pollutants or biomarkers.

[0036] The present invention provides an AAO+MOF chip, which is a cutting-edge sensing device integrating nanotechnology and intelligent materials. It uses anodic aluminum oxide (AAO) nanopore arrays as the structural substrate and integrates a highly active metal-organic framework (MOF) functional layer. Through the precise "pore confinement effect" and "molecular recognition technology", ultrasensitive detection is achieved. Its core structure adopts a multi-layer composite design: the bottom layer is a high-mechanical-strength AAO nanopore array, and the ordered pore structure of AAO significantly increases the effective surface area of the chip, providing an ideal reaction platform for the loading of subsequent functional materials; the middle layer deposits MOF materials as the core unit for signal capture and signal conversion of the chip. The pore structure and surface functional groups of MOF materials can accurately adsorb and recognize a single target substance, avoiding cross-interference, thereby achieving high selectivity for the target analyte. The surface layer is covered with gold nanoparticles, serving as an "amplifier" for signal amplification, converting the adsorption of the target substance into clear electrical or optical signals, and finally achieving high sensitivity and extremely low detection limits for detection. In addition to the advantages in detection performance, it is also remarkable in terms of product reliability.

[0037] The framework stability of the MOF material can complement the high mechanical strength of the AAO nanopore array: AAO can protect the MOF from water molecule erosion through hydrophobic treatment, extending the chip life; while the bonding strength between the metal nodes and organic ligands of the MOF is relatively high and stable, which can cooperate with the Al2O3 layer of AAO to make the chip performance fluctuate less than 5% in the pH range of 2 - 12 and can stably work for more than 3 years in a complex environment, breaking through the bottleneck of traditional chips being vulnerable to humidity interference and having a short life. This chip has now achieved breakthroughs in the fields of environmental monitoring, medical and health, and food safety, and can be further extended to fields such as smart agriculture and aerospace in the future.

[0038] Furthermore, the metal - complex is Cd - DTPA. The analyte is detected by reflection interference spectroscopy, and the target is measured through the change in the interference pattern caused by the optical length difference of the two - interface reflections.

[0039] The chip of the present invention has a high - resolution reflection spectrum. When the specific antibody binds to the analyte, the change in the local refractive index causes a red - shift in the interference spectrum, significantly improving the detection sensitivity. Compared with the planar thin film, the nanoporous thin film has a larger surface area and can bind more receptor molecules, further enhancing the spectral change.

[0040] The fourth technical solution of the present invention: Provide an optical biochip detection system containing the above - mentioned optical biochip.

[0041] The present invention develops a highly sensitive optical biochip to achieve accurate detection of Cd - DTPA, which is beneficial to evaluating the pollution range and severity, and can also provide a scientific basis for formulating effective treatment strategies. Combining advanced detection technologies and multidisciplinary means, it is expected to more efficiently control Cd - DTPA and the environmental pollution problems it causes in the future, and protect the ecosystem and human health.

[0042] The present invention discloses the following technical effects:

[0043] (1) In the optical biochip prepared by the present invention, AAO provides a regular nanopore array with a large specific surface area and a highly uniform pore distribution, providing an ideal carrier for the attachment and growth of ZIF - 8; while ZIF - 8, as a metal - organic framework material, its high porosity and adjustable structure further increase the active sites for biomolecule immobilization, significantly enhancing the sensitivity of the chip and the target capture ability.

[0044] (2) In the optical biochip proposed by the present invention, ZIF - 8 has rich coordination nodes (such as Zn 2+) and active functional groups (such as imidazole groups). Through surface modification, functional groups such as carboxyl groups and amine groups can be easily introduced, which is convenient for immobilizing biomolecules such as antibodies and enzymes. Combining with the mechanical stability of AAO, the functionalized interface can maintain good biocompatibility and chemical stability.

[0045] (3) The Fourier spectrum proposed in the present invention is more suitable for the detection of biochips. The porous framework structure of ZIF-8 and the regular pore array of AAO together provide rich optical and electrochemical signal enhancement paths for the chip. In optical biosensing, the porous structure can amplify the signal change of the reflection interference spectrum, thereby improving the detection limit and achieving precise detection of target substances at ng / L or even lower concentrations.

[0046] (4) The detection method proposed in the present invention combines the physical stability of AAO and the chemical diversity of ZIF-8, and can achieve highly specific detection by simply adjusting the chemical functional layer of the sensing interface.

[0047] (5) The antibody-based optical biochip proposed in the present invention has the advantages of low cost, high stability, short detection time, high detection sensitivity and specificity, strong operability, and is helpful for real-time monitoring of Cd-DTPA. Description of the Drawings

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0049] Figure 1 It is a schematic diagram of the optical biochip detection system for Cd-DTPA;

[0050] Figure 2 It is a schematic diagram of the functionalization treatment of the ZIF-8 thin film;

[0051] Figure 3 Among them, (a) is the spectrogram of the optical biochip before and after adding specific antibodies in Example 1, and (b) is the partial enlarged view of (a);

[0052] Figure 4 Among them, (a) is the spectrogram of the optical biochip before and after adding specific antibodies in Comparative Example 1, and (b) is the spectrogram of the optical biochip before and after adding specific antibodies in Comparative Example 2;

[0053] Figure 5Among them, (a) is the spectrogram of the optical biochip for detecting Pb-DTPA in Example 1 before and after adding the analyte (500 ng / mL of Pb-DPTA); (b) is the spectrogram of the optical biochip for detecting Cd-DTPA in Example 1 before and after adding the analyte (500 ng / mL of Cd-DPTA); (c) is the spectrogram of the optical biochip for detecting Cd-DTPA in Example 1 before and after adding the analyte (1000 ng / mL of Cd-DPTA); (d) is the spectrogram of the optical biochip for detecting Cd-DTPA in Example 1 before and after adding the analyte (2000 ng / mL of Cd-DPTA).

[0054] Figure 6 For (a) is the spectrogram of the optical biochip for detecting Cd-DTPA in Comparative Example 3 before and after adding the analyte (500 ng / mL of Cd-DTPA); (b) is the spectrogram of the optical biochip for detecting Cd-DTPA in Example 1 before and after adding the analyte (500 ng / mL of Cd-DPTA). Detailed implementation manners

[0055] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0056] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0057] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0058] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0059] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0060] The specific antibody of Cd-DTPA used in the following examples and comparative examples was purchased from Sigma-Aldrich, and the product information is as follows: UNSPSC Code: 12352203; eCl@ss: 32160702; NACRES: NA.46.

[0061] Unless otherwise specified, the room temperature involved in the present invention is calculated as 25 ± 5°C.

[0062] Example 1

[0063] A preparation method of an optical biochip for detecting Cd-DTPA:

[0064] (1) Preparation of a regular AAO membrane

[0065] A regular nanoporous AAO membrane was prepared by anodization, and the pore array structure was optimized by combining nanoimprint technology to enhance the specific surface area and regularity of the chip:

[0066] First, high-purity aluminum (purity ≥ 99.99%) was selected as the substrate material and surface pretreatment was carried out. The surface was smoothed by electrochemical polishing. The electrochemical polishing was carried out using a mixed solution of phosphoric acid and ethanol with a volume ratio of 1:4 at a voltage of 20V for 5 minutes to obtain a mirror effect. Subsequently, a positive photoresist with a thickness of 1 μm was uniformly coated on the aluminum surface, and ultraviolet light with a wavelength of 405 nm was used for exposure using a photolithography machine. The exposure energy was 100 mJ / cm 2 , and a photolithography pattern with a regular hole array (hexagonal arrangement with a pore diameter of 200 nm, a pore spacing of 600 nm, and a pore depth of 5 μm) was generated according to the design. After development, the exposed area of aluminum was selectively chemically etched using a 3 wt% phosphoric acid solution for 3 minutes to form a regular initial template. Subsequently, the templated aluminum substrate was placed in an oxalic acid solution (0.3 M) for anodization at a voltage of 50V, a temperature controlled at 5°C, and a time of 1 hour, so that the pore array preferentially expanded along the position of the photolithography template to form a regular nanoporous AAO membrane.

[0067] (2) Uniformly coat the surface of the AAO membrane with zinc-based metal-organic framework material (ZIF-8) using the spin-coating technique

[0068] Dissolve 2-methylimidazole and zinc nitrate hexahydrate in methanol respectively (the concentration of 2-methylimidazole is 0.1 M, and the concentration of zinc nitrate hexahydrate is 0.2 M), and stir well to make them evenly mixed to prepare the ZIF-8 precursor solution. Subsequently, place the prepared AAO membrane on the spin-coating device, spin-coat the ZIF-8 precursor solution at a speed of 2500 rpm for 40 seconds to make the solution evenly cover the surface of the AAO. After spin-coating, place the sample at room temperature and let it stand for drying to promote the uniform growth of ZIF-8 crystals on the surface of the AAO. After drying, a dense and uniformly covered ZIF-8 thin film is formed, providing an ideal functional layer for subsequent biofunctionalization or detection applications.

[0069] (3) Introduce active groups on the surface of the ZIF-8 thin film

[0070] Immerse the ZIF-8 thin film prepared in step (2) in a carboxyl modifier solution (a mixture of terephthalic acid and ethanol with a volume ratio of 1:1), incubate at 50 °C for 30 min, then wash three times with ethanol, and then perform a drying treatment to obtain a functionalized ZIF-8 thin film.

[0071] (4) Covalently immobilize 20 μL of the specific antibody against Cd-DTPA (concentration 1 μM / mL) on the surface of the functionalized ZIF-8 thin film using 40 μL of a cross-linking agent (EDC / NHS). The schematic diagram of the treatment process is shown in Figure 2 , specifically, add EDC / NHS to the ZIF-8 thin film, react at room temperature for 1 hour, and then dropwise add the Cd-DTPA antibody and react at 4 °C for 8 hours to obtain an optical biochip for detecting Cd-DTPA.

[0072] Example 2

[0073] A preparation method of an optical biosensor for detecting Cd-DTPA:

[0074] (1) Prepare a regular AAO membrane

[0075] Prepare a regular nanoporous AAO membrane by anodic oxidation, and combine nanoimprinting technology to optimize the pore array structure and enhance the specific surface area and regularity of the sensor:

[0076] First, prepare the aluminum substrate. Select high-purity aluminum (purity ≥ 99.99%) and make the surface smooth through electrochemical polishing. Use a mixed solution of phosphoric acid and ethanol with a ratio of 1:4 to process at a voltage of 20V for 2 minutes to obtain a mirror effect. Subsequently, uniformly coat a layer of positive photoresist with a thickness of 2μm on the aluminum surface. Use a lithography machine to expose it with ultraviolet light with a wavelength of 405nm, and the exposure energy is 50mJ / cm 2 , and generate a photolithography pattern of a regular hole array according to the design (a hexagonal arrangement with a pore diameter of 500nm, a pore spacing of 1000nm, and a pore depth of 5μm). After development, use a 2wt% phosphoric acid solution to selectively chemically etch the aluminum in the exposed area for 1 minute to form a regular initial template. Subsequently, place the templated aluminum substrate in an oxalic acid solution (0.3M) for anodic oxidation at a voltage of 40V, a temperature controlled at 8°C, and a time of 1.5 hours, so that the pore array preferentially expands along the position of the photolithography template to form a regular nanoporous AAO membrane.

[0077] (2) Use the spin-coating technique to uniformly coat the surface of the AAO membrane with a zinc-based metal-organic framework material (ZIF-8)

[0078] Dissolve 2-methylimidazole and zinc nitrate hexahydrate in methanol respectively (the concentration of 2-methylimidazole is generally 0.1M, and the concentration of zinc nitrate hexahydrate is 0.2M), stir well to make them uniformly mixed, and prepare the ZIF-8 precursor solution. Subsequently, place the prepared AAO membrane on a spin-coating device and spin-coat the ZIF-8 precursor solution at a speed of 2000rpm for a duration of 60 seconds to make the solution uniformly cover the surface of the AAO. After spin-coating, place the sample at room temperature to stand and dry (or dry it in an oven at 50 - 80°C for 10 - 30 minutes) to promote the uniform growth of ZIF-8 crystals on the surface of the AAO. After drying, a dense and uniformly covered ZIF-8 thin film is formed, providing an ideal functional layer for subsequent biofunctionalization or detection applications.

[0079] (3) Introduce active groups on the surface of the ZIF-8 thin film

[0080] Immerse the ZIF-8 thin film prepared in step (2) in a carboxyl modifier solution (a mixture of citric acid and dimethylformamide with a volume ratio of 1:1), incubate at 60°C for 25 minutes, then wash it three times with dimethylformamide, and then perform a drying treatment to obtain a functionalized ZIF-8 thin film.

[0081] (4) Use a cross-linking agent (EDC / NHS) to covalently immobilize the specific antibody against Cd-DTPA on the surface of the functionalized ZIF-8 thin film. The schematic diagram of the treatment process is shown in Figure 2, specifically, EDC / NHS was added to the ZIF-8 film and reacted at room temperature for 1 hour. Then, the Cd-DTPA antibody was added dropwise and reacted at 4 °C for 8 hours to obtain an optical biosensor for detecting Cd-DTPA.

[0082] Example 3

[0083] A preparation method of an optical biosensor for detecting Cd-DTPA:

[0084] (1) Prepare a regular AAO membrane

[0085] A regular nanoporous AAO membrane was prepared by anodization and the pore array structure was optimized by nanoimprinting technology to enhance the specific surface area and regularity of the sensor:

[0086] First, the aluminum substrate was prepared. High-purity aluminum (purity ≥ 99.99%) was selected and the surface was smoothed by mechanical polishing or electrochemical polishing. Electrochemical polishing was carried out using a mixed solution of phosphoric acid and ethanol at a ratio of 1:4 under a voltage of 20 V for 3 minutes to obtain a mirror finish. Subsequently, a positive photoresist with a thickness of 1.5 μm was uniformly coated on the aluminum surface. Exposure was carried out using ultraviolet light with a wavelength of 365 nm by a mask aligner, and the exposure energy was 80 mJ / cm 2 , and a photolithography pattern with a regular pore array (hexagonal arrangement with a pore diameter of 300 nm, a pore spacing of 800 nm, and a pore depth of 5 μm) was generated according to the design. After development, selective chemical etching of the exposed area of aluminum was carried out using a 3 wt% chloride solution for 2 minutes to form a regular initial template. Subsequently, the templated aluminum substrate was placed in an oxalic acid solution (0.3 M) for anodization at a voltage of 60 V, a temperature controlled at 10 °C, and a time of 2 hours, so that the pore array preferentially expanded along the position of the photolithography template to form a regular nanoporous AAO membrane.

[0087] (2) Uniformly coat the zinc-based metal-organic framework material (ZIF-8) on the surface of the AAO membrane by spin coating

[0088] Dissolve 2-methylimidazole and zinc nitrate hexahydrate in methanol respectively (where the concentration of 2-methylimidazole is 0.1 M and the concentration of zinc nitrate hexahydrate is 0.2 M), stir well to make them evenly mixed, and prepare the ZIF-8 precursor solution. Subsequently, place the prepared AAO membrane on a spin-coating device, spin-coat the ZIF-8 precursor solution at a speed of 3000 rpm for 30 seconds to uniformly cover the solution on the surface of AAO. After spin-coating, let the sample stand at room temperature for drying (or dry it in an oven at 50 - 80 °C for 10 - 30 minutes) to promote the uniform growth of ZIF-8 crystals on the surface of AAO. After drying, a dense and uniformly covered ZIF-8 thin film is formed, providing an ideal functional layer for subsequent biofunctionalization or detection applications.

[0089] (3) Introduce active groups on the surface of the ZIF-8 thin film

[0090] Immerse the ZIF-8 thin film prepared in step (2) in a carboxyl modifier solution (a mixture of oxalic acid and deionized water with a volume ratio of 1:1), incubate at 80 °C for 20 min, then wash three times with deionized water, and then perform a drying treatment to obtain a functionalized ZIF-8 thin film.

[0091] (4) Use a cross-linking agent (EDC / NHS) to covalently immobilize the specific antibody against Cd-DTPA on the surface of the functionalized ZIF-8 thin film. The schematic diagram of the treatment process is shown in Figure 2 , specifically add EDC / NHS to the ZIF-8 thin film, react at room temperature for 1 hour, then add the Cd-DTPA antibody dropwise and react at 4 °C for 8 hours to obtain an optical biosensor for detecting Cd-DTPA.

[0092] Comparative Example 1

[0093] The difference from the example is only that ZIF-8 is replaced by MIL-53, and the remaining steps are the same as in Example 1.

[0094] Comparative Example 2

[0095] The difference from the example is only that ZIF-8 is replaced by HKUST-1, and the remaining steps are the same as in Example 1.

[0096] Comparative Example 3

[0097] The difference from the example is only that the treatments in steps (2) and (3) are not performed.

[0098] That is, use a cross-linking agent to immobilize the specific antibody against Cd-DTPA on the surface of AAO.

[0099] The results of the minimum detection limit, antibody immobilization efficiency, and chemical stability of the optical biochips prepared in Examples 1 - 3 and Comparative Examples 1 - 2 of the present invention are shown in Table 1.

[0100] Table 1

[0101]

[0102]

[0103] Among them, the chemical stability was obtained by immersing the optical biochip in a buffer solution with pH = 7.4 and storing it at a constant temperature of 37 °C for 3 days, and analyzing the spectral changes. The calculation formula is as follows:

[0104] Among them:

[0105] I t : The spectral intensity after soaking for t days (t = 3);

[0106] I0: The initial spectral intensity before soaking.

[0107] It can be seen that the optical biochip using ZIF-8 as the porous framework material is significantly superior to MIL-53 and HKUST-1 in terms of detection limit, antibody immobilization efficiency, and chemical stability. This indicates that after replacing with other types of porous framework structure materials, the chip cannot achieve the expected high sensitivity and stability, and cannot meet the accurate detection requirements for target substances at the ng / L level or even lower concentrations.

[0108] Effect verification example

[0109] Based on the optical biochip prepared by the present invention, an optical biochip detection system is constructed to achieve efficient detection of target substances:

[0110] The optical biochip detection system includes a tungsten halogen lamp, an optical fiber probe, a spectrometer, an optical biochip (chip), and a computer; as Figure 1 shown, the broadband light source (300 - 1300 nm) emitted by the tungsten halogen lamp is connected to the optical fiber probe, and the optical fiber probe vertically irradiates the surface of the optical biochip for detecting Cd-DTPA. The reflected signal (conducted signal) is collected by the same optical fiber probe and then imported into the spectrometer (detection range 350 nm - 1050 nm). The spectrometer is connected to a portable computer for data acquisition and processing.

[0111] After dropping the crosslinking agent (EDC / NHS) on the chip prepared in step (3) of Example 1, it is placed in Figure 1 the optical biochip detection system in Figure 1 for spectral detection; then the optical biochip finally prepared in Example 1 is also placed in Figure 3 . Figure 3Among them, (a) is the spectrogram (red line) of the chip component prepared in step (3) of Example 1 after adding the crosslinking agent (EDC / NHS), and the spectrogram of the final prepared optical biochip in Example 1 (blue line); (b) is a partial enlarged view of (a).

[0112] As can be seen from Figure 3 it, compared with the optical chip component without adding specific antibody, the spectral offset of the optical biochip in Example 1 is 1.7 nm.

[0113] After adding the crosslinking agent (EDC / NHS) to the chip component prepared in step (3) of Comparative Example 1, it was placed in Figure 1 the optical biochip detection system in Figure 1 for spectral detection; then the finally prepared optical biochip of Comparative Example 1 was also placed in Figure 4 the optical biochip detection system in

[0114] for spectral detection, and the obtained spectrogram is shown in Figure 1 Figure 1 Figure 4

[0115] (a), where the red line is the spectrogram of the chip component prepared in step (3) of Comparative Example 1 after adding the crosslinking agent (EDC / NHS), and the blue line is the spectrogram of the finally prepared optical biochip of Comparative Example 1.

[0115] As can be seen from Figure 4 (a), compared with the optical chip without adding specific antibody, the spectral offset of the optical biochip for detecting Cd-DTPA prepared in Comparative Example 1 is 0.1 nm.

[0116] As can be seen from Figure 4 (b), compared with the optical chip without adding specific antibody, the spectral offset of the optical biochip for detecting Cd-DTPA prepared in Comparative Example 2 is 0.15 nm.

[0117] Figure 5 Among them, (a) is the optical biochip for detecting Pb-DTPA in Example 1 before and after adding the analyte (500 ng / mL Pb-DPTA) through Figure 1The spectrogram detected by the detection system. Among them, the red line is the spectrogram without the analyte added, and the blue line is the spectrogram after the analyte is added. It can be seen that the spectral offset after adding the analyte is 0.2 nm.

[0118] Figure 5 Among them, (b) is the spectrogram of the optical biochip for detecting Cd-DTPA in Example 1 before and after adding the analyte (500 ng / mL Cd-DPTA) Figure 1 detected by the detection system. Among them, the red line is the spectrogram without the analyte added, and the blue line is the spectrogram after the analyte is added. It can be seen that the spectral offset after adding the analyte is 1.3 nm.

[0119] Figure 5 Among them, (c) is the spectrogram of the optical biochip for detecting Cd-DTPA in Example 1 before and after adding the analyte (1000 ng / mL Cd-DPTA) Figure 1 detected by the detection system. Among them, the red line is the spectrogram without the analyte added, and the blue line is the spectrogram after the analyte is added. It can be seen that the spectral offset after adding the analyte is 2.1 nm.

[0120] Figure 5 Among them, (d) is the spectrogram of the optical biochip for detecting Cd-DTPA in Example 1 before and after adding the analyte (2000 ng / mL Cd-DPTA) Figure 1 detected by the detection system. Among them, the red line is the spectrogram without the analyte added, and the blue line is the spectrogram after the analyte is added. It can be seen that the spectral offset after adding the analyte is 3.3 nm. From Figure 5 it can be seen that the spectrogram of the detection sample containing Cd-DTPA shows an obvious red shift phenomenon, indicating that the optical biochip detection system constructed by the present invention can achieve efficient detection of Cd-DTPA.

[0121] Figure 6 Among them, (a) is the spectrogram of the optical biochip for detecting Cd-DTPA in Comparative Example 3 before and after adding the analyte (500 ng / mL Cd-DTPA); (b) is the spectrogram of the optical biochip for detecting Cd-DTPA in Example 1 before and after adding the analyte (500 ng / mL Cd-DPTA). It can be Figure 6 seen that the chip with ZIF-8 added has a larger offset and higher detection sensitivity when detecting at the same concentration.

[0122] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing an optical biochip, characterized in that, Comprising the following steps: (1) Coating ZIF-8 on the surface of nanoporous alumina to form a ZIF-8 layer on the surface of the nanoporous alumina; (2) Modifying the surface of the ZIF-8 layer with carboxyl or amino groups to obtain a functionalized ZIF-8 layer; (3) Using a crosslinking agent to immobilize a specific antibody on the surface of the functionalized ZIF-8 layer to obtain the optical biochip.

2. The preparation method of the optical biochip according to claim 1, characterized in that, The nanoporous alumina has a regular pore array structure.

3. The preparation method of the optical biochip according to claim 2, wherein, The preparation method of the nanoporous alumina comprises the following steps: First, designing a regular pore array pattern on the surface of an aluminum substrate, then obtaining an aluminum substrate template with a regular pore array through chemical etching, and finally performing anodic oxidation on it to obtain the nanoporous alumina.

4. The preparation method of the optical biochip according to claim 3, characterized in that The solution used for the anodic oxidation is an oxalic acid solution; the anodic oxidation voltage is 40 - 60V, and the temperature is 5 - 10°C.

5. The preparation method of the optical biochip according to claim 1, characterized in that in step (2), carboxyl modification is carried out using a carboxyl modifier; the carboxyl modifier is terephthalic acid, citric acid or oxalic acid; in step (2), amino modification is carried out using an amino modifier; the amino modifier is ethylenediamine or 1,6-hexanediamine.

6. The preparation method of the optical biochip according to claim 5, wherein, The specific antibody is a Cd-DTPA specific antibody.

7. An optical biochip prepared by the preparation method according to any one of claims 1 - 6.

8. The application of the optical biochip according to claim 7 in the detection of metal-complexes, environmental pollutants or biomarkers.

9. The application according to claim 8, characterized in that, The metal-complex is Cd-DTPA.

10. An optical biochip detection system, characterized in that Containing the optical biochip according to claim 7.

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