Repeatable functionalization method for inner wall of microcavity

Abstract

The invention relates to a method for repeatedly functionalizing a microcavity. Repeatable functionalization of the inner wall of the microcavity is realized by adopting an extra-cavity magnetic bead dynamic modification technology. The method comprises the following steps: carrying out surface modification by adopting ferroferric oxide magnetic beads, and modifying specific antibodies or other specific substances on the surfaces of the magnetic beads; introducing magnetic beads into the microcavity through a microfluidic system, and applying an external magnetic field to enable the magnetic beads to be adsorbed on the inner wall of the microcavity so as to capture the to-be-detected substance; after the detection is completed, the magnetic beads are removed by utilizing the microfluidic equipment, so that the microcavity is recovered to the initial state and can be repeatedly used. The method has the characteristics of simplicity and convenience in operation, wide application range and reusability, and is suitable for unmarked detection, such as biological detection, gas detection and the like, in which the microcavity needs to be specifically modified.

Description

Technical Field

[0001] This invention relates to the field of sensing technology, specifically to a method for reproducible functionalization of the inner wall of a microcavity, applicable to achieving highly sensitive and reproducible functionalization in optical microcavity sensing systems. Background Technology

[0002] Label-free optical sensing technology identifies and analyzes target substances by monitoring changes in physical quantities, such as refractive index, absorption coefficient, or scattering intensity, caused by the interaction between the analyte and the sensing interface. This type of method eliminates the need for complex labels such as fluorescent markers, enzyme reactions, or dyes, offering advantages such as real-time performance and ease of operation. It is widely used in environmental monitoring, gas detection, industrial process control, food safety, and chemical analysis.

[0003] Although this type of technology has good application prospects, it often relies on large and complex precision instruments, and the detection system has low integration, making it difficult to meet the needs of portable or high-throughput on-site detection, which limits its promotion in practical application scenarios.

[0004] With the development of micro-nano photonics, novel sensing systems based on integrated optical devices are gradually emerging. Among them, optical microcavities have shown significant advantages in various label-free detection applications due to their small size, high quality factor, and strong sensitivity.

[0005] Optical microcavities are a typical type of high-quality factor optical structure. They couple with the target material through an evanescent field and can respond to extremely small changes in the refractive index of the cavity surface, thereby enabling sensitive detection of trace substances such as gas molecules, volatile organic compounds, particulate matter, or biomolecules.

[0006] To achieve microcavity detection, a functionalized layer with specific recognition capabilities is typically constructed on the inner wall of the microcavity. However, traditional chemical modification methods often employ covalent bonding to immobilize the trapped molecules, which is not only cumbersome and irreversible, making it difficult to reuse the microcavity, but may also cause irreversible damage to the optical properties of the cavity.

[0007] Therefore, there is an urgent need for a microcavity interface construction method that is simple in structure, controllable in operation, and supports reversible functionalization, so as to improve the practicality and flexibility of optical microcavities in label-free detection in multiple fields. Summary of the Invention

[0008] The purpose of this invention is to address the difficulties in functionalizing the inner wall of microcavities and the fact that functionalization can only be performed once, by proposing a method for repeatable functionalization.

[0009] This invention proposes a method for reproducible functionalization of the inner wall of a microcavity. This method involves functionalizing the magnetic beads by modifying the surface of carboxyl magnetic beads with specific substances, transferring the magnetic beads into the microcavity using a microfluidic system, and then adsorbing the magnetic beads onto the inner wall of the microcavity using an electromagnet probe.

[0010] This invention includes a microcavity, an electromagnet probe, carboxyl magnetic beads, and a microfluidic system. The specific steps of the invention are as follows: (1) Passing the carboxyl-containing magnetic beads into an activator to activate the carboxyl groups on the surface of the magnetic beads; (2) Quickly mixing the magnetic beads with a solution containing specific materials and allowing the reaction to stand for 2 hours; (3) Cleaning the inner wall of the microcavity; (4) Using the microfluidic system to pass the magnetic beads into the microcavity, and after the flow rate stabilizes, turning on the external magnetic field to attract the magnetic beads to the inner wall of the microcavity; (5) Using the microfluidic system to flush away excess unadsorbed magnetic beads.

[0011] The equipment used in this invention includes tunable lasers, fiber optic fusion splicers, microfluidic pumps, and DC electromagnets.

[0012] The magnetic beads mentioned in step (1) of this invention have a diameter of 50 nm to 2.5 μm and are mainly composed of iron(III) oxide, and the surface of the magnetic beads has been modified with carboxyl groups. The activator is generally an EDC and NHS solution, and the activation time is about 30 min.

[0013] The specific materials in step (2) of this invention include, but are not limited to, protein molecules or DNA molecules, such as antibodies and aptamers.

[0014] The magnitude of the external magnetic field applied in step (4) of this invention needs to be appropriate, so that the inner wall of the microcavity can adsorb a single layer or a small number of magnetic beads; otherwise, the excessive number of magnetic beads will cause difficulties in subsequent sensing.

[0015] Compared with traditional methods of functionalizing the inner wall of microcavities, the present invention has the following advantages.

[0016] (1) Simple operation: Compared with traditional microcavity functionalization, traditional microcavity functionalization requires chemical modification or molecular connection inside the cavity, which is complicated and difficult to control. However, this invention simplifies the operation process by functionalizing magnetic beads outside the cavity, and introduces the functionalized magnetic beads into the microcavity through an external magnetic field, avoiding direct chemical treatment of the microcavity and protecting the integrity of the microcavity.

[0017] (2) Repeatability: Since the functionalization process of magnetic beads is reversible, after a test is completed, the magnetic beads attached to the inner wall of the microcavity can be removed by microfluidic flushing to restore the initial state and facilitate reuse. This repeatability not only reduces the experimental cost, but also reduces the complexity of experimental operation.

[0018] (3) High flexibility: By using functionalized magnetic beads with different surface modifications and using an external magnetic field to control the adsorption and release of the magnetic beads, different functionalization modes can be quickly switched to achieve specific detection of multiple target substances in the same microcavity.

[0019] (4) High compatibility: The method of the present invention is compatible with a variety of functionalized magnetic beads and detection targets, including but not limited to the detection of a variety of biomolecules such as exosomes, proteins, and nucleic acids, as well as other detections that require specific modification in the microcavity, such as the specific detection of gases. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the operation of the present invention.

[0021] Figure 2 This is a schematic diagram of the system structure of the present invention.

[0022] Figure 3 This is a schematic diagram of the process for specific detection of microcavity surface modifications.

[0023] Figure 4 The transmission spectra of the magnetic beads before and after they adhere to the wall.

[0024] Figure 5 A schematic diagram illustrating the signal changes caused by capturing the analyte.

[0025] The numbers in the diagram are as follows: 1 is a tunable laser; 2 is a microcavity; 3 is a single-mode fiber optic cone; 4 is an oscilloscope; 5 is a computer; 6 is a microfluidic device; 7 is a waste liquid tank; 8 is an optical path; and 9 is a microfluidic channel. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to the accompanying drawings. The overall operation flow of the present invention is as follows: Figure 1 flow chart.

[0027] The present invention includes a single-mode fiber taper, a microcavity, an electromagnet probe, a carboxyl magnetic bead, and a microfluidic system.

[0028] The main component of the fiber taper is silicon dioxide, with a diameter of 1-2 μm, which is used to couple light into and out of the microcavity.

[0029] The main component of the microcavity is silicon dioxide, with a diameter of 60-300 μm and a wall thickness of 1-5 μm.

[0030] An electromagnet probe is used to provide an external magnetic field, with the magnetic field strength provided by the probe tip being greater than 100 mT.

[0031] Carboxylated magnetic beads are mainly composed of iron(III) oxide and have a diameter of 2.5 μm, serving as functionalized carriers.

[0032] The microfluidic system is connected to both ends of the microcavity to introduce solution into the microcavity. A schematic diagram of the system structure of this invention is shown below. Figure 2 As shown.

[0033] Mix the magnetic beads and add freshly prepared EDC and NHS solutions into a centrifuge tube containing the magnetic beads. Vortex to mix and fully suspend the magnetic beads. Activate at 25°C for 30 minutes to activate the carboxyl groups on the surface of the magnetic beads.

[0034] Antibody was added to the magnetic bead solution and mixed well. After reacting at 25°C for 2 hours, the supernatant was removed by magnetic separation. The beads were washed with PBS solution (pH 7.2) each time. The functionalized magnetic beads were obtained.

[0035] A microfluidic system was used to introduce PBS solution (pH 7.2) into the microcavity to clean the inner wall of the microcavity and create a liquid environment.

[0036] The magnetic bead solution was mixed and diluted with PBS solution (pH 7.2) and then introduced into the microcavity. After the flow rate stabilized, the electromagnet probe switch was turned on, attracting the magnetic beads to the inner wall of the microcavity. The functionalization process is as follows: Figure 3 As shown.

[0037] After rinsing with PBS solution (pH 7.2) to remove excess unadsorbed magnetic beads, a shift in the resonant wavelength and mode broadening of the transmission spectrum can be observed before the magnetic beads adhere to the microcavity. Figure 4 As shown.

[0038] Pass a solution containing the substance to be tested through the solution and let it stand for 1 hour to allow it to fully react with the binding sites on the magnetic beads.

[0039] Rinse with PBS solution (pH 7.2) to remove any residual solution.

[0040] After processing, the acquired signal can be used to obtain the signal changes caused by the analyte, mainly manifested as a shift in the center wavelength caused by changes in refractive index, such as... Figure 5 As shown.

[0041] Turn off the electromagnet probe and flush with PBS solution (pH 7.2) to restore the microcavity to its original state for reuse.

[0042] The examples of this invention are preferred embodiments, but are not limited thereto. Any embodiment that does not depart from the spirit of this invention is within the scope of protection of this invention.

Claims

1. A method for reproducibly functionalizing the inner wall of a microcavity, characterized in that, Includes the following steps: (a) The surface of carboxyl-modified iron oxide magnetic beads is activated outside the cavity to fix the specific material onto the surface of the magnetic beads and obtain functionalized magnetic beads. (b) Functionalized magnetic beads are injected into the microcavity and the magnetic beads are adsorbed onto the inner wall surface of the microcavity by applying an external magnetic field; (c) Remove the unadsorbed magnetic beads to complete the functionalization process of the inner wall of the microcavity; (d) After the test is completed, the adsorbed magnetic beads are removed by the microfluidic system to restore the microcavity to its initial state so that it can be reused.

2. The method according to claim 1, characterized in that, The magnetic beads are carboxyl-modified iron oxide magnetic beads with a diameter of 50 nm to 2.5 μm. The surface of the magnetic beads is activated by EDC and NHS solutions for 20-40 min.

3. The method according to claim 1, characterized in that, The specific material is selected from antibodies, aptamers, or other specific substances.

4. The method according to claim 1, characterized in that, The external magnetic field is provided by an electromagnet probe, and the duration of the magnetic field action is matched with the injection flow rate of the magnetic beads to achieve the adsorption of single-layer or near-single-layer magnetic beads on the inner wall of the microcavity.

5. A sensing system for implementing the method according to claims 1-4, characterized in that, The microcavity is connected to the microfluidic channel; an electromagnet probe, located outside the microcavity, is used to provide a controllable magnetic field; functionalized magnetic beads contain a variety of surface-modified specific materials; and an optical detection module, including a tunable laser, a fiber taper, and an oscilloscope, is used to excite optical signals and acquire detection data.

6. The system according to claim 5, characterized in that, The flow rate of the microfluidic channel is adjustable in the range of 1-50 μl / min, and the diameter of the cone region of the fiber taper is 1-2 μm.

7. The system according to claim 5, characterized in that, The magnetic field strength of the electromagnet probe can be adjusted between 10-200 mT, and the distance between the probe tip and the outer wall of the microcavity is 10-100 μm.

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