DNA biosensor based on reflection interference spectrum, preparation method and application of DNA biosensor in detection of doxorubicin

Abstract

The invention discloses a DNA biosensor based on a reflection interference spectrum, a preparation method and application of the DNA biosensor to detection of doxorubicin. The DNA biosensor is characterized in that uniformly distributed nanopores are processed in the upper surface of an anodic aluminum oxide substrate, a copper layer is arranged on the upper surface of the anodic aluminum oxide substrate outside the nanopores, probe ssDNA is connected to the walls of the nanopores through Schiff base bonds, and target ssDNA is connected to the probe ssDNA. According to the present invention, the optical signal change during the process of filling the drug molecules into the DNA molecules can be monitored in real time through the reflection interference spectrum technology, the long-time incubation and washing steps are not required, the detection time is substantially shortened, the rapid detection requirement is met, and the timely detection result is provided especially for the doxorubicin detection application.

Description

Technical Field

[0001] The present invention belongs to the technical field of testing by measuring chemical or physical properties of materials, and particularly relates to a DNA biosensor based on reflection interference spectroscopy, a preparation method thereof, and an application for detecting doxorubicin. Background Art

[0002] The study of the interaction between DNA and small molecule drugs is a key link in the research and development of anti-tumor drugs. As a commonly used anti-cancer drug, accurate detection of the concentration of doxorubicin is of great significance for efficacy evaluation and drug optimization.

[0003] Currently, the commonly used detection methods for doxorubicin include fluorescence labeling method, ultraviolet detection technology, Raman detection technology, and electrochemical method. The fluorescence labeling method requires a labeling agent, and the labeling agent molecules will affect the experimental results. The ultraviolet detection technology has limited sensitivity. At low concentrations, it may not be able to accurately detect trace amounts of doxorubicin, is easily interfered by other substances with similar ultraviolet absorption characteristics, and has high requirements for the purity of samples. The Raman detection technology has high requirements for equipment, requires high laser power and sensitive detectors, has high requirements for the detection scenario and the surrounding environment, and for substances that produce fluorescence, the detection process will overlap with the Raman signal. The electrochemical method has insufficient specificity. The redox reaction occurring on the electrode surface will interfere with the specific detection of doxorubicin, and changes in factors such as the ionic strength, pH value, temperature, and dissolved oxygen of the solution for electrochemical detection will all affect the electrochemical signal.

[0004] Therefore, the existing detection methods have problems such as insufficient sensitivity, specificity, and stability, and it is difficult to meet the needs of precision medicine and drug research and development. At the same time, there is a lack of efficient means for the detection of specific drugs such as doxorubicin. There is an urgent need for a drug detection technology with high sensitivity, high stability, and high speed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a DNA biosensor based on reflection interference spectroscopy and a preparation method thereof with high sensitivity, good stability, and fast detection speed.

[0006] The technical solution adopted to solve the above technical problem is: A DNA biosensor based on reflection interference spectroscopy, wherein the upper surface of an anodic aluminum oxide substrate is processed with uniformly distributed nanopores, a copper layer is provided on the upper surface of the anodic aluminum oxide substrate outside the nanopores, a probe ssDNA is connected to the pore wall of the nanopores through Schiff base bonds, and a target ssDNA is connected to the probe ssDNA.

[0007] Preferably, the aperture of the nanopores is 79 nm to 85 nm, and the pore depth is 7.4 μm to 7.8 μm.

[0008] Preferably, the thickness of the copper layer is 10 nm to 20 nm.

[0009] A preparation method of a DNA biosensor based on reflection interference spectroscopy, comprising the following steps:

[0010] Step 1. Prepare a nanoporous anodic aluminum oxide substrate;

[0011] Step 2. Coat a copper layer on the surface of the nanoporous anodic aluminum oxide substrate by atomic layer deposition to form a copper-coated nanoporous anodic aluminum oxide substrate;

[0012] Step 3. Put the copper-coated nanoporous anodic aluminum oxide substrate into a hydrogen peroxide solution and boil it, then cool and wash it to hydroxylate the surface of the nanopore walls of the copper-coated nanoporous anodic aluminum oxide substrate;

[0013] Step 4. Immerse the copper-coated nanoporous anodic aluminum oxide substrate in a silane coupling agent solution for reaction to form an aminated surface on the surface of the nanopore walls, and then wash and dry it;

[0014] Step 5. Immerse the copper-coated nanoporous anodic aluminum oxide substrate treated in Step 4 in a glutaraldehyde solution for reaction to generate Schiff base bonds on the surface of the nanopore walls;

[0015] Step 6. Drop 5'-amino-modified ssDNA onto the copper-coated nanoporous anodic aluminum oxide substrate treated in Step 5 and react in an environment with humidity > 90% to form covalent bonds between -NH2 of ssDNA and -CHO on the pore walls, thereby forming probe ssDNA on the surface of the nanopore walls;

[0016] Step 7. Wash the surface of the substrate with 1 mol / L NaCl solution to remove unreacted 5'-amino-modified ssDNA;

[0017] Step 8. Drop 10 -5 mol / L of n-propylamine onto the upper surface of the substrate, let it stand at room temperature for a period of time, and then wash and dry it;

[0018] Step 9. Drop the target ssDNA onto the upper surface of the substrate, let it stand at a constant temperature for a period of time to allow the target ssDNA to hybridize fully with the probe ssDNA, and complete the fabrication of the DNA biosensor.

[0019] Preferably, the mass concentration of the silane coupling agent in the silane coupling agent solution is 5%.

[0020] Preferably, the reaction temperature in Step 4 is room temperature, and the reaction time is 50 min to 70 min;

[0021] The cleaning and drying method in step 4 is as follows: First, immerse the copper-plated nanoporous anodic aluminum oxide substrate in an ethanol solution to remove the unreacted silane coupling agent on the surface, then thoroughly rinse it with clear water, and place it in a vacuum drying oven at 45 °C for drying for 30 min.

[0022] Preferably, the mass concentration of glutaraldehyde in the glutaraldehyde solution is 25%, and the pH is 7.4.

[0023] Preferably, the cleaning in step 8 is to slowly rinse with deionized water.

[0024] A DNA biosensor based on reflection interference spectroscopy and its application in detecting doxorubicin.

[0025] The beneficial effects of the present invention are as follows:

[0026] 1. The present invention utilizes light irradiation on the nanopore structure of the anodic aluminum oxide substrate, which can form a reflection interference spectrum with characteristic wavelengths. The reflection interference spectrum is extremely sensitive to changes in the optical properties of the nanostructure surface. When drug molecules are filled between the target ssDNA and the probe ssDNA, the refractive index in the nanopore channel changes significantly. Especially when a copper layer is provided on the surface of the anodic aluminum oxide substrate, the light field intensity is significantly enhanced, so that tiny changes can be accurately detected through the reflection interference spectrum, realizing high-sensitivity monitoring of DNA specific binding. The detection limit can reach the femtomole level, and it has higher sensitivity compared with traditional optical detection methods.

[0027] 2. The present invention firmly connects the probe ssDNA to the surface of the nanopore wall through Schiff base bonds. This covalent bond connection method ensures the stable fixation and correct orientation of the probe ssDNA, which is beneficial to the specific binding between the target ssDNA and the probe ssDNA.

[0028] 3. The detection process of the present invention is simple. Since the reflection interference spectroscopy technology can monitor the change of optical signals during the process of drug molecules filling into DNA molecules in real time, without long incubation and washing steps, the detection time is greatly shortened, meeting the need for rapid detection, and providing timely detection results for application scenarios such as clinical diagnosis and drug research and development.

[0029] 4. The anodic aluminum oxide substrate of the present invention has good chemical stability and mechanical stability, and can maintain the integrity of its nanopore structure under different experimental environments and operating conditions. The deposition of the copper layer further enhances the conductivity and corrosion resistance of the substrate, improving the overall stability of the DNA biosensor. In addition, through chemical modification means such as silane coupling agent and glutaraldehyde, the probe ssDNA is stably fixed on the surface of the nanopore wall, reducing the shedding and denaturation of DNA molecules during the detection process, and ensuring the performance stability of the DNA biosensor after multiple uses and long-term storage. Description of the Drawings

[0030] Figure 1 is a schematic diagram of the structure of the DNA biosensor based on reflectance interference spectroscopy of the present invention.

[0031] Figure 2 is a schematic diagram of the fixing process of the probe ssDNA and the target ssDNA of the DNA biosensor based on reflectance interference spectroscopy of the present invention.

[0032] Figure 3 is a schematic diagram of doxorubicin molecules intercalating into the reflection of the target ssDNA and the probe ssDNA.

[0033] Figure 4 is the reflectance interference spectrum and EOT diagram measured on the copper-plated nanoporous anodic aluminum oxide substrate of the present invention.

[0034] Figure 5 is the reflectance interference spectrum and EOT diagram measured by the DNA biosensor based on reflectance interference spectroscopy of the present invention.

[0035] Figure 6 is the reflectance interference spectrum and the effective optical thickness EOT diagram detected after adding doxorubicin solution to the sensor of the present invention. Detailed Description of the Invention

[0036] The present invention will be further described in detail below with reference to the drawings and embodiments, but the present invention is not limited to the following embodiments.

[0037] In Figure 1 the DNA biosensor based on reflectance interference spectroscopy of this embodiment, the upper surface of the anodic aluminum oxide substrate is processed with uniformly distributed nanopores. The thickness of the anodic aluminum oxide substrate is 0.5 mm. The pore diameter of the nanopores is 79 nm to 85 nm, and the pore depth is 7.4 μm to 7.8 μm. A copper layer is provided on the upper surface of the anodic aluminum oxide substrate outside the nanopores, and the thickness of the copper layer is 10 nm to 20 nm. The probe ssDNA is connected to the pore wall of the nanopores through Schiff base bonds, and the target ssDNA is connected to the probe ssDNA.

[0038] When light irradiates the surface of the anodic aluminum oxide substrate with a nanopore structure, due to the special structure of the nanopores, light undergoes multiple reflections and interferences on the inner pore walls. This interference phenomenon causes constructive or destructive interference of light with specific wavelengths, and thus forms a reflection spectrum with characteristic wavelengths. The copper layer outside the nanopores can generate a local surface plasmon resonance effect. When light irradiates the copper layer, the free electrons in the copper layer interact with the light to produce a resonance phenomenon. This resonance effect can further enhance the local light field intensity, making the characteristic peaks of the reflection spectrum more prominent, thereby enhancing the intensity of the detection signal.

[0039] When a drug molecule binds to the target DNA and the probe ssDNA, it causes a change in the refractive index of the nanopore wall. This change in refractive index affects the propagation and interference of light within the nanopore, thereby causing a shift in the characteristic peak of the reflection spectrum or a change in intensity.

[0040] A method for preparing a DNA biosensor based on reflection interference spectroscopy according to this embodiment includes the following steps:

[0041] Step 1. Prepare a nanoporous anodic aluminum oxide substrate with a substrate thickness of 0.5 mm, a nanopore diameter of 79 nm to 85 nm, and a pore depth of 7.4 μm to 7.8 μm;

[0042] The specific preparation method of the nanoporous anodic aluminum oxide substrate is a known technique and is disclosed in "Study on the Sensitivity of Reflection Interference Spectroscopy Technology Based on Nanoporous Anodic Aluminum Oxide Sensing Substrate" published in Acta Photonica Sinica on September 18, 2024.

[0043] Step 2. Coat a copper layer on the surface of the nanoporous anodic aluminum oxide substrate by atomic layer deposition. The thickness of the copper layer is 10 nm to 20 nm, and in this embodiment, it is 15 nm, to form a copper-coated nanoporous anodic aluminum oxide substrate;

[0044] Step 3. Place the copper-coated nanoporous anodic aluminum oxide substrate in a boiling hydrogen peroxide solution and keep it for 30 min to hydroxylate the surface of the nanopore wall of the copper-coated nanoporous anodic aluminum oxide substrate, then cool it to room temperature, place it in deionized water, and gently shake it for 15 min for washing;

[0045] Step 4. Immerse the copper-coated nanoporous anodic aluminum oxide substrate in a silane coupling agent solution and react for 1 hour to form an aminated surface on the surface of the nanopore wall as an organic modification layer. Among them, the silane coupling agent solution is an anhydrous ethanol solution with a mass concentration of 2% of the silane coupling agent. Then, immerse the copper-coated nanoporous anodic aluminum oxide substrate in an ethanol solution to remove the unreacted silane coupling agent on the surface, then thoroughly rinse it with water, and place it in a vacuum drying oven at 45 °C for drying for 30 min;

[0046] Step 5. Immerse the copper-coated nanoporous anodic aluminum oxide substrate treated in Step 4 in a glutaraldehyde solution and react for 12 hours. During the reaction, the aldehyde group (-CHO) of glutaraldehyde reacts with the amino group (-NH2) in the organic modification layer to generate Schiff base bonds on the surface of the nanopore wall, providing reaction sites for the subsequent immobilization of DNA molecules. After the reaction, dry it with nitrogen to prevent molecular inactivation caused by moisture;

[0047] Step 6. Drop 20 μL of 5'-amino-modified ssDNA solution with 45 base pairs onto the copper-plated nanoporous anodic aluminum oxide substrate processed in Step 5, and react for 12 hours in an environment with humidity > 90%, so that -NH2 of ssDNA forms a covalent bond with -CHO on the pore wall, thereby forming probe ssDNA on the surface of the nanopore wall;

[0048] Step 7. Wash the substrate surface with 1 mol / L NaCl solution to remove the unreacted 5'-amino-modified ssDNA;

[0049] Step 8. Drop 10 -5 mol / L of n-propylamine onto the substrate surface to improve the complementary pairing efficiency of target ssDNA and probe ssDNA. After standing at room temperature for 6 hours, wash and dry;

[0050] Step 9. Drop 20 μL of target ssDNA with 45 base pairs at a concentration of 9.8×10 -2 mol / L onto the upper surface of the substrate, and let it stand at a constant temperature for a period of time to allow the target ssDNA to hybridize fully with the probe ssDNA. For example, Figure 2 , thus completing the fabrication of the DNA biosensor.

[0051] The DNA biosensor based on reflection interference spectroscopy in this example is used to detect doxorubicin. The specific detection method is as follows:

[0052] Place the DNA biosensor based on reflection interference spectroscopy on the translation stage of the reflection interference spectroscopy detection system, and detect that the original effective optical thickness value of this DNA biosensor is 3.525;

[0053] Drop 20 μL of doxorubicin solution at a concentration of 1.2×10 -5 mol / L onto the DNA biosensor based on reflection interference spectroscopy, and let it stand for 2 hours to allow doxorubicin molecules to embed between the target ssDNA and the probe ssDNA. Then detect through the reflection interference spectroscopy detection system, and the obtained effective optical thickness value is 3.615, and this result > the original effective optical thickness value.

[0054] The principle is as follows: As doxorubicin molecules embed between the target ssDNA and the probe ssDNA, for example, Figure 3 , the substances in the nanopore channels are increased. The effective optical thickness value EOT = 2nL, where n is the refractive index and L is the substrate thickness. As the substances (drug molecules) filling the pores increase, the refractive index also becomes larger, resulting in an increase in the effective optical thickness value.

[0055] To verify the beneficial effects of the present invention, during the preparation of the DNA biosensor based on reflection interference spectroscopy, the inventors used a reflection interference spectroscopy detection system to sequentially detect the copper-plated nanoporous anodic aluminum oxide substrate, the DNA biosensor of the present invention, and the reflection interference spectroscopy and effective optical thickness EOT after adding doxorubicin solution to the sensor of the present invention, as Figures 4 - 6 .

[0056] It can be seen therefrom that by observing the change in the value of the effective optical thickness EOT, the present invention can intuitively judge whether there is an interaction between DNA and drug molecules and the degree of the interaction.

Claims

1. A DNA biosensor based on reflection interference spectroscopy, characterized in that: The upper surface of the anodized aluminum oxide substrate is processed with uniformly distributed nanopores. A copper layer is provided on the upper surface of the anodized aluminum oxide substrate outside the nanopores. Probe ssDNA is connected to the pore wall of the nanopores through Schiff base bonds, and target ssDNA is connected to the probe ssDNA.

2. The DNA biosensor based on reflection interference spectroscopy according to claim 1, wherein: The aperture of the nanopores is 79 nm to 85 nm, and the pore depth is 7.4 μm to 7.8 μm.

3. The DNA biosensor based on reflectance interference spectroscopy according to claim 1, characterized in that: The thickness of the copper layer is 10 nm to 20 nm.

4. A preparation method of a DNA biosensor based on reflection interference spectroscopy, characterized in that, It includes the following steps: Step 1. Prepare a nanoporous anodized aluminum oxide substrate. Step 2. Cover a copper layer on the surface of the nanoporous anodized aluminum oxide substrate by atomic layer deposition to form a copper-plated nanoporous anodized aluminum oxide substrate. Step 3. After boiling the copper-plated nanoporous anodized aluminum oxide substrate in a hydrogen peroxide solution, then cool and wash it to hydroxylate the surface of the pore wall of the copper-plated nanoporous anodized aluminum oxide substrate. Step 4. Immerse the copper-plated nanoporous anodized aluminum oxide substrate in a silane coupling agent solution for reaction to form an aminated surface on the surface of the pore wall of the nanopores, and then wash and dry it. Step 5. Immerse the copper-plated nanoporous anodized aluminum oxide substrate treated in Step 4 in a glutaraldehyde solution for reaction to generate Schiff base bonds on the surface of the pore wall of the nanopores. Step 6. Drop 5'-amino-modified ssDNA onto the copper-plated nanoporous anodized aluminum oxide substrate treated in Step 5 and react in an environment with humidity > 90% to form a covalent bond between -NH2 of ssDNA and -CHO on the pore wall, so as to form probe ssDNA on the surface of the pore wall of the nanopores. Step 7. Wash the surface of the substrate with 1 mol / L NaCl solution to remove the unreacted 5'-amino-modified ssDNA. Step 8. Drop 10 -5 mol / L of n-propylamine onto the upper surface of the substrate, let it stand at room temperature for a period of time, then wash and dry; Step 9. Drop target ssDNA onto the upper surface of the substrate, and let it stand at a constant temperature for a period of time to allow the target ssDNA to hybridize fully with the probe ssDNA, thus completing the fabrication of the DNA biosensor.

5. The method according to claim 4, wherein The mass concentration of the silane coupling agent in the silane coupling agent solution is 5%.

6. The method according to claim 4, wherein The reaction temperature in Step 4 is room temperature, and the reaction time is 50 min to 70 min. The washing and drying method in Step 4 is as follows: First, immerse the copper-plated nanoporous anodized aluminum oxide substrate in an ethanol solution to remove the unreacted silane coupling agent on the surface, then rinse it thoroughly with water, and place it in a vacuum drying oven at 45 °C for drying for 30 min.

7. The method according to claim 4, characterized in that The mass concentration of glutaraldehyde in the glutaraldehyde solution is 25%, and the pH is 7.

4.

8. The method according to claim 4, wherein The washing in Step 8 is to slowly rinse with deionized water.

9. A DNA biosensor based on reflection interference spectroscopy according to any one of claims 1 to 3, characterized in that: Application in detecting doxorubicin.

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