A highly sensitive optical biochip based on an AAO and ZIF-8 composite structure and its fabrication and detection method

Abstract

This invention discloses a highly sensitive optical biochip based on an AAO and ZIF-8 composite structure, and its preparation and detection method, belonging to the field of optical biochip detection technology. In this invention, ZIF-8 is coated onto the surface of nanoporous alumina to obtain a ZIF-8 layer. Then, carboxyl or amino group modification is performed to obtain a functionalized ZIF-8 layer. Subsequently, a crosslinking agent is used to immobilize specific antibodies on the surface of the functionalized ZIF-8 layer, resulting in the optical biosensor. This sensor uses an AAO nanopore array as the structural substrate and integrates a highly active MOF functional layer. It achieves ultrasensitive detection through precise "pore confinement effect" and "molecular recognition technology." The optical biosensor prepared by this invention has advantages such as low cost, high stability, short detection time, and high detection sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the field of optical biochip detection technology, and in particular to a highly sensitive optical biochip based on an AAO and ZIF-8 composite structure, and its preparation and detection methods. Background Technology

[0002] Cd-DTPA is cadmium ion (Cd) 2+ The stable chelate formed by Cd and diethylenetriaminepentaacetic acid (DTPA) is a common metal-organic complex widely found in industrial wastewater and mining drainage. DTPA, as a highly efficient chelating agent, can form a stable polydentate coordination structure with cadmium ions, thereby significantly increasing the solubility and migration of cadmium. This property makes Cd-DTPA play an important role in the environment: on the one hand, its formation may reduce free Cd. 2+ On the one hand, it has direct toxicity; on the other hand, it enhances the migration ability of cadmium, enabling it to spread over long distances and infiltrate groundwater systems, causing secondary pollution and significantly increasing the difficulty of environmental remediation.

[0003] The environmental hazards of Cd-DTPA mainly lie in its long-term stability and potential toxicity. Although chelated cadmium exhibits low biotoxicity in certain situations, Cd-DTPA may dissociate in acidic or strongly oxidizing environments, releasing highly toxic free Cd. 2+ Cd-DTPA poses a serious threat to aquatic and soil ecosystems. Furthermore, it can contaminate groundwater through infiltration or runoff, affecting drinking water safety, and enter the food chain through bioaccumulation, causing long-term harm to human and animal health. Cadmium is a known carcinogen, and excessive exposure can lead to kidney damage, bone disease, and other health problems. The stable presence of Cd-DTPA makes traditional pollution control technologies (such as precipitation or adsorption) ineffective in removing cadmium, further exacerbating the difficulty of its remediation.

[0004] Currently, there are diverse technical methods for detecting Cd-DTPA, mainly including spectroscopic analysis, electrochemical detection, and chromatographic techniques. Spectroscopic methods (such as UV-Vis absorption spectroscopy and atomic absorption spectroscopy) identify Cd-DTPA by measuring absorbance or luminescence intensity at specific wavelengths; electrochemical methods (such as anodic stripping voltammetry) detect Cd-DTPA by measuring changes in current signals; and liquid chromatography (such as high-performance liquid chromatography, HPLC) combined with mass spectrometry can achieve highly sensitive analysis of Cd-DTPA. In addition, nanotechnology, such as nanopore sensors and reflectance interference spectroscopy (RIfS), which has been developed in recent years, has also become a potential tool for Cd-DTPA detection due to its rapid response and high sensitivity. The detection range of different methods is typically between μg / L and ng / L, with emerging nanotechnology achieving even lower detection limits, making it suitable for trace pollutant monitoring.

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

[0006] The purpose of this invention is to provide a highly sensitive optical biochip based on a nanoporous alumina (AAO) and ZIF-8 composite structure, along with its preparation and detection method, to address the problems existing in the prior art. This optical biochip possesses advantages such as low cost, high stability, short detection time, and high detection sensitivity and specificity, providing technical support for the rapid detection of complex heavy metal complexes.

[0007] This invention constructs a highly efficient sensing interface by combining a metal-organic framework material (ZIF-8) with regularly spaced nanoporous anolyl alumina (AAO), thus preparing an optical biochip for detecting Cd-DTPA. The detection of Cd-DTPA is achieved using reflectance interferometry (RIfS). This optical biochip not only fully utilizes the structural regularity of the AAO membrane and the high specific surface area of ​​ZIF-8, but also improves antibody immobilization efficiency and target analyte capture ability through surface functionalization, providing technical support for the rapid detection of complex heavy metal complexes.

[0008] This invention provides the following solution:

[0009] One of the technical solutions of this invention is to provide a method for preparing an optical biochip, comprising the following steps:

[0010] (1) Coating ZIF-8 onto 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 amino groups to obtain a functionalized ZIF-8 layer;

[0012] (3) The specific antibody is immobilized on the surface of the functionalized ZIF-8 layer using a cross-linking 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 nanoporous alumina with a regular pore array structure used in this invention preferably has a pore size between 150-500 nm and a pore depth between 15-20 μm. More 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] A regular pore array pattern is designed on the surface of an aluminum substrate, and chemical etching is performed to obtain an aluminum substrate template with a regular pore array. Then, anodizing is performed to obtain the nanoporous aluminum oxide.

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

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

[0020] More specifically, the preparation process of nanoporous alumina is as follows: First, high-purity aluminum (purity ≥ 99.99%) is selected as the substrate material and surface pretreatment is performed. The surface is smoothed by mechanical polishing or electrochemical polishing. Electrochemical polishing typically uses an acid solution treated at 20V for 2-5 minutes to obtain a mirror effect. Subsequently, a layer of positive photoresist with a thickness of 1-2μm is uniformly coated on the aluminum surface, and exposure is performed using a photolithography machine with ultraviolet light at a wavelength of 405nm or 365nm, and the exposure energy is 50-100mJ / cm². 2 A photolithographic pattern of a regular hole array is generated according to the design. After development, the exposed aluminum area is selectively chemically etched with a 1-3 wt% phosphoric acid solution or chloride solution for 1-3 minutes to form a regular initial template. Subsequently, the templated aluminum substrate is placed in an oxalic acid solution (0.3M) for anodizing at a voltage of 40-60V and a temperature controlled at 5-10℃ for 1-2 hours, allowing the hole array to preferentially expand along the position of the photolithographic template, forming a regular nanopore AAO.

[0021] The more specific preparation process of the functional layer is as follows: A ZIF-8 precursor solution is prepared by dissolving 2-methylimidazole and zinc nitrate hexahydrate in methanol, typically at a concentration of 0.1M to 0.2M, and stirring thoroughly to ensure homogeneity. Subsequently, the prepared AAO is placed on a spin-coating device, and the ZIF-8 precursor solution is spin-coated at 2000-3000 rpm for 30-60 seconds to ensure uniform coverage of the AAO surface. After spin-coating, the sample is allowed to dry at room temperature or in an oven at 50-80℃ for 10-30 minutes to promote uniform growth of ZIF-8 crystals on the AAO surface. After drying, a dense and uniformly covered ZIF-8 film is formed, providing an ideal functional layer for subsequent biofunctionalization or detection applications.

[0022] Furthermore, in step (2), a carboxyl group is modified using a carboxyl group modifier, wherein the carboxyl group modifier is terephthalic acid (TPA), citric acid, or oxalic acid;

[0023] The carboxyl modifier used in this invention has multiple carboxyl groups, which can undergo coordination reactions with metal nodes (such as Zn2+) on the ZIF-8 surface.

[0024] Further, the modifier is dissolved in a suitable solvent, such as ethanol, dimethylformamide (DMF), or deionized water, to form a homogeneous reaction solution. Subsequently, the ZIF-8 material is immersed in the modifier solution and incubated at room temperature or under appropriate heating conditions (e.g., 50-80°C) for a period of time (preferably 30 min) to promote the binding of the carboxyl modifier to the ZIF-8 surface.

[0025] In step (2), an amine modifier is used for amine modification; the amine modifier may be ethylenediamine or 1,6-hexanediamine.

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

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

[0028] Compared to ZIF-8, other types of porous framework materials (such as MIL-53, HKUST-1, etc.) exhibit significant disadvantages in the application of this invention. The specific reasons are as follows:

[0029] 1) Insufficient aperture and pore volume

[0030] The aperture of ZIF-8 is approximately ZIF-8 can effectively capture and immobilize target molecules. Compared with other porous materials, its pore size and pore volume characteristics are more suitable for the high-sensitivity biochip developed in this invention. For example, HKUST-1 has a larger pore size (approximately...). This results in insufficient selectivity and capture ability for small molecules, making it impossible to effectively amplify signal changes in the reflection interference spectrum.

[0031] 2) Low 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 exhibits poor stability in aqueous solutions and is prone to structural collapse, leading to a significant decrease in the signal enhancement effect of the chip. In contrast, the ZIF-8 material used in this invention demonstrates excellent thermal and chemical stability under various environmental conditions, ensuring the long-term reliability of the chip.

[0033] 3) Convenience of functional processing

[0034] ZIF-8 possesses abundant coordination nodes (such as Zn2+) and active functional groups (such as imidazole groups), allowing for the easy introduction of functional groups such as carboxyl and amine groups through surface modification, facilitating the immobilization of biomolecules such as antibodies and enzymes. In contrast, other porous materials often require more complex chemical modification steps during functionalization, and the modified materials do not exhibit the same biomolecule immobilization efficiency and stability as the ZIF-8 material used in this invention.

[0035] The third technical solution of the present invention is to provide the application of the above-mentioned optical biochip in the detection of metal-coordination compounds, environmental pollutants or biomarkers.

[0036] This invention provides an AAO+MOF chip, a cutting-edge sensing device integrating nanotechnology and smart materials. It uses an anodic aluminum oxide (AAO) nanopore array as its structural substrate, integrating a highly active metal-organic framework (MOF) functional layer to achieve ultrasensitive detection through precise "pore confinement effect" and "molecular recognition technology." Its core structure employs a multi-layered composite design: the bottom layer is a high-mechanical-strength AAO nanopore array; the ordered pore structure of AAO significantly increases the chip's effective surface area, providing an ideal reaction platform for the loading of subsequent functional materials. The middle layer deposits MOF material as the core unit for signal capture and signal conversion; the pore structure and surface functional groups of the MOF material can accurately adsorb and identify single target substances, avoiding cross-interference, thus achieving high selectivity for the target analyte. The surface layer is covered with gold nanoparticles, acting as an "amplifier" to convert the adsorption of the target analyte into a clear electrical or optical signal, ultimately achieving high sensitivity and extremely low detection limit. In addition to its advantages in detection performance, its product reliability is also remarkable.

[0037] The framework stability of MOF materials complements the high mechanical strength of AAO nanopore arrays: AAO, through hydrophobic treatment, protects MOF from water molecule corrosion, extending chip lifespan; while the high bonding strength and stability between the metal nodes and organic ligands of MOF, which synergizes with the Al2O3 layer of AAO, ensures that the chip's performance fluctuation is <5% within a pH range of 2-12, allowing it to operate stably for more than 3 years in complex environments, overcoming the bottlenecks of traditional chips being susceptible to humidity interference and having short lifespans. This chip has already achieved breakthroughs in environmental monitoring, healthcare, and food safety, and can be further extended to smart agriculture, aerospace, and other fields.

[0038] Furthermore, the metal-complex is Cd-DTPA. The analyte is detected using reflection interferometry, which measures the target object by analyzing the changes in the interference pattern caused by the difference in optical lengths reflected from the two interfaces.

[0039] The chip of this invention has a high-resolution reflectance spectrum. When a specific antibody binds to an analyte, the change in local refractive index causes a redshift in the interference spectrum, significantly improving detection sensitivity. Compared to planar thin films, nanoporous thin films have a larger surface area, enabling them to bind more receptor molecules and further enhancing spectral changes.

[0040] The fourth technical solution of the present invention provides an optical biochip detection system, which includes the above-mentioned optical biochip.

[0041] This invention develops a highly sensitive optical biochip to achieve accurate detection of Cd-DTPA, which is beneficial for assessing the scope and severity of pollution and can also provide a scientific basis for formulating effective remediation strategies. Combined with advanced detection technology and multidisciplinary methods, it is expected to more efficiently control Cd-DTPA and the environmental pollution problems it causes in the future, and protect ecosystems 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 highly uniform pore distribution, which provides an ideal carrier for the attachment and growth of ZIF-8; while ZIF-8, as a metal-organic framework material, has high porosity and tunable structure, which further increases the active sites for biomolecule fixation, significantly improving the chip's sensitivity and target capture ability.

[0044] (2) In the optical biochip proposed in this invention, ZIF-8 has abundant coordination nodes (such as Zn). 2+It contains active functional groups (such as imidazole groups), and through surface modification, functional groups such as carboxyl and amine groups can be easily introduced, which facilitates the immobilization of biomolecules such as antibodies and enzymes. Combined with the mechanical stability of AAO, the functionalized interface can maintain good biocompatibility and chemical stability.

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

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

[0047] (5) The antibody-based optical biochip proposed in this 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. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 A schematic diagram of an optical biochip detection system for Cd-DTPA;

[0050] Figure 2 A schematic diagram of the functionalization process of ZIF-8 thin films;

[0051] Figure 3 In the image, (a) shows the spectrum of the optical biochip in Example 1 before and after the addition of the specific antibody, and (b) is a magnified view of (a).

[0052] Figure 4 In the image, (a) shows the spectra of the optical biochip of Comparative Example 1 before and after the addition of the specific antibody, and (b) shows the spectra of the optical biochip of Comparative Example 2 before and after the addition of the specific antibody.

[0053] Figure 5In the figures, (a) shows the spectra of the optical biochip used for detecting Pb-DTPA in Example 1 before and after the addition of the analyte (500 ng / mL Pb-DPTA); (b) shows the spectra of the optical biochip used for detecting Cd-DTPA in Example 1 before and after the addition of the analyte (500 ng / mL Cd-DPTA); (c) shows the spectra of the optical biochip used for detecting Cd-DTPA in Example 1 before and after the addition of the analyte (1000 ng / mL Cd-DPTA); and (d) shows the spectra of the optical biochip used for detecting Cd-DTPA in Example 1 before and after the addition of the analyte (2000 ng / mL Cd-DPTA).

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

[0055] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0056] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0057] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0058] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0059] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0060] The specific antibodies against Cd-DTPA used in the following examples and comparative examples were 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 this invention is 25±5℃.

[0062] Example 1

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

[0064] (1) Preparation of regular AAO membranes

[0065] Regularly shaped nanoporous AAO membranes were prepared by anodic oxidation, and the pore array structure was optimized by nanoimprinting 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 subjected to surface pretreatment. The surface was smoothed by electrochemical polishing using a 1:4 volume ratio mixture of phosphoric acid and ethanol at 20V for 5 minutes to achieve a mirror-like finish. Subsequently, a 1μm thick layer of positive photoresist was uniformly coated onto the aluminum surface, and exposure was performed using a photolithography machine with 405nm wavelength ultraviolet light at an exposure energy of 100mJ / cm². 2 A photolithographic pattern of a regular array of holes (hexagonal arrangement with a hole diameter of 200 nm, a hole spacing of 600 nm, and a hole depth of 5 μm) was generated according to the design. After development, the exposed aluminum area was selectively chemically etched with 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 anodizing at a voltage of 50 V and a temperature controlled at 5 °C for 1 hour, allowing the hole array to preferentially expand along the position of the photolithographic template to form a regular nanoporous AAO film.

[0067] (2) Zinc-based metal-organic framework material (ZIF-8) was uniformly coated onto the surface of the AAO membrane using spin coating technology.

[0068] 2-Methylimidazole and zinc nitrate hexahydrate were dissolved separately in methanol (2-methylimidazole concentration: 0.1 M, zinc nitrate hexahydrate concentration: 0.2 M), and thoroughly stirred to obtain a homogeneous ZIF-8 precursor solution. Subsequently, the prepared AAO membrane was placed on a spin-coating device, and the ZIF-8 precursor solution was spin-coated at 2500 rpm for 40 seconds to ensure uniform coverage of the AAO surface. After spin-coating, the sample was allowed to dry at room temperature to promote uniform growth of ZIF-8 crystals on the AAO surface. After drying, a dense and uniformly covered ZIF-8 film was formed, providing an ideal functional layer for subsequent biofunctionalization or detection applications.

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

[0070] The ZIF-8 film prepared in step (2) was immersed in a carboxyl modifier solution (a mixture of terephthalic acid and ethanol in a volume ratio of 1:1), incubated at 50°C for 30 min, then washed three times with ethanol, and then dried to obtain the functionalized ZIF-8 film.

[0071] (4) A schematic diagram of the surface treatment process of covalently immobilizing 20 μL of anti-Cd-DTPA specific antibody (concentration 1 μm / mL) on a functionalized ZIF-8 film using 40 μL of crosslinking agent (EDC / NHS) is shown below. Figure 2 Specifically, EDC / NHS is added to a ZIF-8 membrane and reacted at room temperature for 1 hour. Then, Cd-DTPA antibody is added and reacted at 4°C for 8 hours to obtain an optical biochip for detecting Cd-DTPA.

[0072] Example 2

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

[0074] (1) Preparation of regular AAO membranes

[0075] Regularly shaped nanoporous AAO membranes were prepared by anodic oxidation, and the pore array structure was optimized by nanoimprinting technology to enhance the specific surface area and regularity of the sensor.

[0076] First, the aluminum substrate is prepared using high-purity aluminum (purity ≥ 99.99%). The surface is smoothed by electrochemical polishing using a 1:4 mixture of phosphoric acid and ethanol at 20V for 2 minutes to achieve a mirror finish. Subsequently, a 2μm thick layer of positive photoresist is uniformly coated onto the aluminum surface, and the surface is exposed to 405nm ultraviolet light using a photolithography machine at an exposure energy of 50mJ / cm². 2 A photolithographic pattern of a regular array of holes (hexagonal arrangement with a hole diameter of 500 nm, a hole spacing of 1000 nm, and a hole depth of 5 μm) was generated according to the design. After development, the exposed aluminum area was selectively chemically etched with a 2 wt% phosphoric acid solution for 1 minute to form a regular initial template. Subsequently, the templated aluminum substrate was placed in an oxalic acid solution (0.3 M) for anodizing at a voltage of 40 V and a temperature controlled at 8 °C for 1.5 hours, allowing the hole array to preferentially expand along the position of the photolithographic template to form a regular nanoporous AAO film.

[0077] (2) Zinc-based metal-organic framework material (ZIF-8) was uniformly coated onto the surface of the AAO membrane using spin coating technology.

[0078] 2-Methylimidazole and zinc nitrate hexahydrate were dissolved separately in methanol (2-methylimidazole concentration: 0.1 M, zinc nitrate hexahydrate concentration: 0.2 M), and thoroughly stirred to obtain a homogeneous ZIF-8 precursor solution. Subsequently, the prepared AAO membrane was placed on a spin-coating device, and the ZIF-8 precursor solution was spin-coated at 2000 rpm for 60 seconds to ensure uniform coverage of the AAO surface. After spin-coating, the sample was allowed to dry at room temperature (or in an oven at 50-80℃ for 10-30 minutes) to promote uniform growth of ZIF-8 crystals on the AAO surface. After drying, a dense and uniformly covered ZIF-8 film was formed, providing an ideal functional layer for subsequent biofunctionalization or detection applications.

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

[0080] The ZIF-8 film prepared in step (2) was immersed in a carboxyl modification agent solution (a mixture of citric acid and dimethylformamide in a volume ratio of 1:1), incubated at 60°C for 25 min, then washed three times with dimethylformamide, and then dried to obtain the functionalized ZIF-8 film.

[0081] (4) A schematic diagram of the process of covalently immobilizing the specific antibody against Cd-DTPA on the surface of a functionalized ZIF-8 film using a crosslinking agent (EDC / NHS) is shown below. Figure 2Specifically, EDC / NHS was added to the ZIF-8 membrane and reacted at room temperature for 1 hour. Then, Cd-DTPA antibody was added and reacted at 4°C for 8 hours to obtain an optical biosensor for detecting Cd-DTPA.

[0082] Example 3

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

[0084] (1) Preparation of regular AAO membranes

[0085] Regularly shaped nanoporous AAO membranes were prepared by anodic oxidation, 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 is prepared using high-purity aluminum (purity ≥ 99.99%). The surface is smoothed by mechanical or electrochemical polishing. Electrochemical polishing uses a 1:4 mixture of phosphoric acid and ethanol at 20V for 3 minutes to achieve a mirror finish. Then, a 1.5μm thick layer of positive photoresist is uniformly coated onto the aluminum surface, and exposed using ultraviolet light at a wavelength of 365nm with an exposure energy of 80mJ / cm². 2 A photolithographic pattern of a regular array of holes (hexagonal arrangement with a hole diameter of 300 nm, a hole spacing of 800 nm, and a hole depth of 5 μm) was generated according to the design. After development, the exposed aluminum area was selectively chemically etched 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 anodizing at a voltage of 60 V and a temperature controlled at 10 °C for 2 hours, allowing the hole array to preferentially expand along the position of the photolithographic template to form a regular nanoporous AAO film.

[0087] (2) Zinc-based metal-organic framework material (ZIF-8) was uniformly coated onto the surface of the AAO membrane using spin coating technology.

[0088] 2-Methylimidazole and zinc nitrate hexahydrate were dissolved separately in methanol (2-methylimidazole concentration: 0.1 M, zinc nitrate hexahydrate concentration: 0.2 M), and thoroughly stirred to obtain a homogeneous ZIF-8 precursor solution. Subsequently, the prepared AAO membrane was placed on a spin-coating device, and the ZIF-8 precursor solution was spin-coated at 3000 rpm for 30 seconds to ensure uniform coverage of the AAO surface. After spin-coating, the sample was allowed to dry at room temperature (or in an oven at 50-80℃ for 10-30 minutes) to promote uniform growth of ZIF-8 crystals on the AAO surface. After drying, a dense and uniformly covered ZIF-8 film was formed, providing an ideal functional layer for subsequent biofunctionalization or detection applications.

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

[0090] The ZIF-8 film prepared in step (2) was immersed in a carboxyl modifier solution (a mixture of oxalic acid and deionized water in a volume ratio of 1:1), incubated at 80°C for 20 min, then washed three times with deionized water, and then dried to obtain the functionalized ZIF-8 film.

[0091] (4) A schematic diagram of the process of covalently immobilizing the specific antibody against Cd-DTPA on the surface of a functionalized ZIF-8 film using a crosslinking agent (EDC / NHS) is shown below. Figure 2 Specifically, EDC / NHS was added to the ZIF-8 membrane and reacted at room temperature for 1 hour. Then, Cd-DTPA antibody was added and reacted at 4°C for 8 hours to obtain an optical biosensor for detecting Cd-DTPA.

[0092] Comparative Example 1

[0093] The only difference from the embodiment is that ZIF-8 is replaced with MIL-53, and the rest of the steps are the same as in embodiment 1.

[0094] Comparative Example 2

[0095] The only difference from the embodiment is that ZIF-8 is replaced with HKUST-1, and the rest of the steps are the same as in embodiment 1.

[0096] Comparative Example 3

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

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

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

[0100] Table 1

[0101]

[0102]

[0103] Chemical stability was determined by immersing the optical biochip in a buffer solution at pH 7.4 and storing it at 37°C for 3 days, followed by analysis of spectral changes. The calculation formula is as follows:

[0104] in:

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

[0106] I0: Initial spectral intensity before immersion.

[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 it with other types of porous framework materials, the chip cannot achieve the expected high sensitivity and stability, and cannot meet the requirements for accurate detection of target analytes at the ng / L level or even lower.

[0108] Effect verification example

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

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

[0111] After adding the crosslinking agent (EDC / NHS) to the chip prepared in step (3) of Example 1, it was placed in... Figure 1 Spectral detection was performed using the optical biochip detection system in the example; then, the optical biochip finally prepared in Example 1 was also placed in the system. Figure 1 The spectral analysis was performed using the optical biochip detection system in the image, and the resulting spectrum is shown below. Figure 3 . Figure 3In the image, (a) is the spectrum of the chip prepared in step (3) of Example 1 after adding crosslinking agent (EDC / NHS) (red line) and the spectrum of the optical biochip finally prepared in Example 1 (blue line); (b) is a magnified view of (a).

[0112] from Figure 3 As can be seen, compared to optical chips without added specific antibodies, the optical biochip in Example 1 exhibits a spectral shift of 1.7 nm.

[0113] After adding the crosslinking agent (EDC / NHS) to the chip prepared in step (3) of Comparative Example 1, it was placed in... Figure 1 Spectral detection was performed using the optical biochip detection system in the example; then, the optical biochip finally prepared in Comparative Example 1 was also placed in the same system. Figure 1 The spectral analysis was performed using the optical biochip detection system in the image, and the resulting spectrum is shown below. Figure 4 (a), where the red line is the spectrum of the chip prepared in step (3) of Comparative Example 1 after adding crosslinking agent (EDC / NHS), and the blue line is the spectrum of the optical biochip finally prepared in Comparative Example 1.

[0114] After adding the crosslinking agent (EDC / NHS) to the chip prepared in step (3) of Comparative Example 2, it was placed in... Figure 1 Spectral detection was performed using the optical biochip detection system in Comparative Example 2; subsequently, the optical biochip finally prepared in Comparative Example 2 was also placed in the system. Figure 1 The spectral analysis was performed using the optical biochip detection system in the image, and the resulting spectrum is shown below. Figure 4 (b), where the red line is the spectrum of the chip prepared in step (3) of Comparative Example 2 after adding crosslinking agent (EDC / NHS), and the blue line is the spectrum of the optical biochip finally prepared in Comparative Example 2.

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

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

[0117] Figure 5 In Example 1, (a) shows the optical biochip used to detect Pb-DTTA before and after the addition of the analyte (500 ng / mL Pb-DPTA). Figure 1The detection system obtained the spectrum, where the red line represents the spectrum without the analyte, and the blue line represents the spectrum with the analyte added. It can be seen that the spectral shift after adding the analyte is 0.2 nm.

[0118] Figure 5 In Example 1, (b) shows the optical biochip used for detecting Cd-DTTA before and after the addition of the analyte (500 ng / mL Cd-DPTA). Figure 1 The detection system obtained the following spectra, where the red line represents the spectrum without the analyte, and the blue line represents the spectrum with the analyte added. It can be seen that the spectral shift after adding the analyte is 1.3 nm.

[0119] Figure 5 In Example 1, (c) shows the optical biochip used to detect Cd-DTPA before and after the addition of the analyte (1000 ng / mL Cd-DPTA). Figure 1 The detection system obtained the following spectra, where the red line represents the spectrum without the analyte, and the blue line represents the spectrum with the analyte added. It can be seen that the spectral shift after adding the analyte is 2.1 nm.

[0120] Figure 5 In the image, (d) shows the optical biochip used in Example 1 for detecting Cd-DTPA before and after the addition of the analyte (2000 ng / mL Cd-DPTA). Figure 1 The detection system obtained the following spectra, where the red line represents the spectrum without the analyte, and the blue line represents the spectrum with the analyte added. It can be seen that the spectral shift after adding the analyte is 3.3 nm. From... Figure 5 As can be seen, the spectrum of the detection sample containing Cd-DTPA shows a significant red shift, indicating that the optical biochip detection system constructed in this invention can achieve efficient detection of Cd-DTPA.

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

[0122] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for fabricating an optical biochip, characterized in that, Includes the following steps: (1) Coating ZIF-8 on the surface of nanoporous alumina to form a ZIF-8 layer on the surface of nanoporous alumina; (2) Modify the surface of the ZIF-8 layer with carboxyl or amino groups to obtain a functionalized ZIF-8 layer; (3) The specific antibody is immobilized on the surface of the functionalized ZIF-8 layer using a cross-linking agent to obtain the optical biochip; The nanoporous alumina has a regular pore array structure; The preparation method of the nanoporous alumina includes the following steps: First, a regular pore array pattern is designed on the surface of an aluminum substrate. Then, an aluminum substrate template with a regular pore array is obtained by chemical etching. Finally, it is anodized to obtain the nanoporous aluminum oxide. The anodizing solution used is oxalic acid solution; the anodizing voltage is 40-60V and the temperature is 5-10℃. In step (2), carboxyl modification is performed using a carboxyl modifying agent; the carboxyl modifying agent is terephthalic acid, citric acid, or oxalic acid. In step (2), an amine group is modified using an amine group modifier; the amine group modifier is ethylenediamine or 1,6-hexanediamine; The specific antibody is a Cd-DTPA specific antibody.

2. The optical biochip prepared by the method described in claim 1.

3. The application of the optical biochip as described in claim 2 in the detection of metal-complexes, environmental pollutants or biomarkers.

4. The application according to claim 3, characterized in that, The metal-complex is Cd-DTPA.

5. An optical biochip detection system, characterized in that, It contains the optical biochip as described in claim 2.

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