DNA (deoxyribonucleic acid) modified titanium dioxide film sensor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biosensors, and particularly discloses a DNA-modified titanium dioxide film sensor as well as a preparation method and application thereof. The method comprises the following steps: plating gold on a titanium dioxide nanotube prepared by an anodic oxidation method, modifying a DNA probe under the action of a gold sulfhydryl bond, obtaining electrons by taking a platinum electrode as a cathode and losing electrons by taking metal titanium as an anode in an electrolyte in an oxidation process, and oxidizing under external voltage. Due to the existence of the nano pore channels, under the illumination condition, photon-generated carriers can directionally migrate along the surface potential in the direction of the pore channels and drive ions in the pore channels to directionally move; the specific recognition effect of the surface probe and the target causes the change of the surface charge and changes the surface potential, so that a measurable electric signal is generated, and universal target detection and multi-target simultaneous in-situ detection can be realized. The method provided by the invention has great significance in early detection, screening and treatment of prostate cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to a DNA-modified titanium dioxide thin film sensor and a preparation method and application thereof. Background Art

[0002] Rapid, highly sensitive detection of biomolecules is crucial for disease diagnosis. While accurate, traditional detection methods (such as high-performance liquid chromatography and mass spectrometry) suffer from drawbacks such as expensive equipment, complex procedures, and time-consuming procedures, making them difficult to meet the demands of point-of-care (POCT) testing. Consequently, the development of low-cost, highly sensitive, and selective biosensors has become a research hotspot.

[0003] Electrochemical biosensors are widely used for the detection of small biomolecules due to their fast response, ease of operation, and amenability to miniaturization. The material selection for the sensing interface is crucial to sensor performance. Titanium dioxide (TiO2), a wide-bandgap semiconductor material, exhibits excellent chemical stability, biocompatibility, superior photoelectric properties, and high catalytic activity, and has been used to construct a variety of biosensors. However, the poor conductivity of pure TiO2 films and their limited selectivity for biomolecule detection limit their further application in biosensing.

[0004] In recent years, researchers have optimized the properties of TiO2 thin films through methods such as doping (such as nitrogen doping and carbon doping) and composites (such as combining with gold nanoparticles, graphene, and carbon nanotubes) to improve their conductivity and ability to immobilize biomolecules. Furthermore, surface modification with biorecognition elements such as enzymes, antibodies, or aptamers can significantly enhance the detection selectivity and sensitivity of TiO2-based sensors for specific molecules. For example, immobilizing glucose oxidase (GOx) on the surface of TiO2 nanostructures can achieve efficient electrocatalytic oxidation of glucose, thereby constructing a highly sensitive glucose sensor. However, existing TiO2-based biosensors still face problems such as insufficient stability, easy enzyme inactivation, and low interfacial electron transfer efficiency, and further optimization of material structure and sensing strategies is urgently needed.

[0005] Therefore, developing a biosensor based on a new type of titanium dioxide film to improve the detection performance of biomolecules through the combination of material modification, interface optimization and biomolecule immobilization technology has important scientific research value and practical application prospects. Summary of the Invention

[0006] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a DNA-modified titanium dioxide thin film sensor and its preparation method and application, which can achieve efficient capture and detection of specific biomolecules, thereby realizing early diagnosis.

[0007] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention is to provide a method for preparing a DNA-modified titanium dioxide thin film sensor, comprising the following steps: S1, anodizing the titanium sheet into a titanium dioxide nanotube array, A two-electrode system is used, with a titanium sheet as the working electrode and a platinum sheet as the auxiliary electrode. A voltage of 20-25 V is applied to oxidize the surface of the titanium sheet for 30-90 minutes to form a titanium dioxide nanotube array. S2, spraying the prepared titanium dioxide nanotubes with gold in a gold plating apparatus; S3. Immerse the gold-sprayed titanium dioxide in a thiol-modified DNA solution for modification, thereby obtaining the DNA-modified titanium dioxide thin film sensor; the DNA is an aptamer of the biomolecule to be detected.

[0008] Furthermore, in step S2, the thickness of the gold spraying is 10-30 nm.

[0009] The second object of the present invention is to provide a titanium dioxide thin film sensor prepared by the above-mentioned preparation method.

[0010] The third object of the present invention is to provide the use of the above-mentioned titanium dioxide thin film sensor in detecting biological molecules.

[0011] A fourth objective of the present invention is to provide a device for detecting biological molecules, comprising two chambers, wherein the above-mentioned titanium dioxide thin film sensor is arranged between the two chambers, an ultraviolet light source is arranged above the titanium dioxide thin film sensor, and both ends of the titanium dioxide thin film sensor are connected to the two chambers respectively. Both chambers are filled with potassium chloride solution and are respectively provided with a working electrode and a reference electrode. The detection of biological small molecules is achieved by recording the transmembrane ion current before and after the addition of the biological molecule sample to be tested.

[0012] Furthermore, the wavelength of the ultraviolet light source is 364 nm, and the intensity range is 82.5~121.5 mW / cm 2 .

[0013] Furthermore, the concentration of the potassium chloride solution is not less than 1 μM / L.

[0014] A fifth object of the present invention is to provide a method for detecting markers for early diagnosis of prostate cancer, using the above-mentioned device and a picoammeter to measure the transmembrane ion current before and after the addition of the test sample, and to calculate the current change rate, using the formula: Current change rate % = (I - I0) / I0, Where I0 is the current value measured before the sample to be tested is added, and I is the current value measured after the sample to be tested is added.

[0015] Furthermore, the diagnostic marker is sarcosine or PSA protein.

[0016] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: (1) Due to the presence of nanopores, the titanium dioxide thin film sensor provided by the present invention can, under illumination conditions, cause photogenerated carriers to migrate directionally along the surface potential in the direction of the pores, and drive the ions in the pores to move in a directional manner. The specific recognition of the surface probe and target causes changes in the surface charge and changes in the surface potential, thereby generating a measurable electrical signal. At the same time, the confined space on the surface of the nanopores can enhance the target-probe collision and binding efficiency, thereby improving the recognition of the probe and target. The photoelectrocatalysis of the nanoarray can improve the conversion efficiency and conduction efficiency of chemical signals to electrical signals due to the efficient mass transfer of the confined pores, thereby achieving improved sensitivity. The photoelectrocatalysis of the nanoarray can reduce the impact of environmental changes due to the highly controlled nanoscale reaction space, thereby achieving improved selectivity. The photoelectrocatalysis of the nanoarray can be modified by partitioning and functionalization, which can achieve universal target detection and simultaneous in-situ detection of multiple targets.

[0017] (2) The present invention gold-plates titanium dioxide nanotubes prepared by an anodic oxidation method, and modifies DNA probes corresponding to PSA proteins or sarcosine small molecules under the action of gold-sulfhydryl bonds. During the oxidation process, the platinum electrode acts as a cathode to gain electrons in the electrolyte, and the metal titanium acts as an anode to lose electrons, and oxidizes under an applied voltage. On the one hand, the surface charge change in the channel is used for judgment under an applied voltage. On the other hand, the photoelectric effect of titanium dioxide is used to generate electron-hole separation under the irradiation of an external xenon lamp to achieve cancer detection. The current value before and after antibody binding is used to preliminarily judge the cancer status, which is of great significance for the early detection, screening and treatment of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Comparison of the microscopic morphologies of titanium dioxide nanotubes anodized at different voltages. In the figure, AD is the SEM image of the front side of the titanium dioxide nanotube, and EH is the SEM image of the back side of the titanium dioxide nanotube. Figure 2 Comparison of the morphologies of titanium dioxide nanotubes prepared under different anodization time conditions. AE in the figure is the SEM image of the front side of the titanium dioxide nanotube, and FJ is the SEM image of the back side of the titanium dioxide nanotube. Figure 3 The figure shows the change of ion transmission performance before and after sputtering gold. In the figure, A is the external voltage and external pressure ion current (IV), and B is the light energy ion current (It); Figure 4 This is a graph showing the cyclic stability of titanium dioxide nanotubes provided by the present invention; Figure 5 The comparison diagram of contact angle changes before and after modification of probe and target; Figure 6 This is a graph showing the effect of different concentrations of the cancer small molecule marker sarcosine detected in Examples 1 to 3 of the present invention under the same other conditions, demonstrating that there is a significant effect on the response to different concentrations of sarcosine; Figure 7 Graph showing the effect of light intensity on cancer detection in Example 1, Examples 4, and 5 of the present invention under the same other conditions, showing that light intensity is substantially positively correlated with photocurrent. Figure 8 This is a graph showing the effect of applying ultraviolet radiation on cancer detection in Example 1 and Example 6 of the present invention under the same other conditions. The photoelectric effect is used to amplify the signal and improve the detection sensitivity. Figure 9 This is the effect of changing the potassium chloride concentration on cancer detection under the same other conditions as in Example 1 of the present invention; Figure 10 Example 1 of the present invention uses DNA to detect different cancer marker proteins under the same conditions to prove the specific effect on prostate cancer; Figure 11 This is a diagram showing the specific effects on prostate cancer demonstrated by using DNA to detect different cancer small molecule markers under the same conditions as in Example 1 of the present invention; Figure 12 This is a graph showing the change in specific response signals of patients and healthy subjects using DNA to detect real blood samples under the same conditions in Example 1 of the present invention. Compared with healthy subjects, patients have obvious current fluctuations and a greater signal change rate. DETAILED DESCRIPTION

[0019] To make the objects, technical solutions, and advantages of the present invention more apparent, the following describes the specific embodiments of the present invention in further detail with reference to the specific examples and accompanying drawings. Where specific test methods, instruments, or conditions are not specified in the examples, the methods or conditions described in the literature in the art or the product specifications were used. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0020] Example 1 The present invention provides a method for preparing a DNA-modified titanium dioxide thin film sensor, which comprises the following steps: Step S1, pretreatment of titanium sheet; Cut the titanium sheet into a square shape with a length of 1.5 cm and a width of 1 cm; The titanium sheet was initially cleaned in concentrated hydrochloric acid to remove surface stains, and then rinsed several times with deionized water; The cleaned titanium sheet is clamped on the working electrode of the electroplating tank, and the platinum sheet is used as the auxiliary electrode; Step S2, anodizing the titanium sheet into a titanium dioxide nanotube array; Specifically, S201, preparing an electrolyte required for anodization, which is prepared according to 50 ml of water, 450 ml of ethylene glycol and 2.75425 g of ammonium fluoride; S202, clamping the cleaned titanium sheet on the working electrode of the electroplating tank and the platinum sheet as the auxiliary electrode, with the two metal sheets facing each other; S203, turning on the power supply and adjusting the voltage to (10 V, 15 V, 20 V, 25 V) to oxidize the surface of the titanium sheet to form titanium dioxide nanotubes. The oxidation time is (30 min, 45 min, 60 min, 75 min, 90 min); S204, S203 oxidized titanium dioxide was rinsed in deionized water for 3 min; Step S3, spraying gold on the titanium dioxide nanotubes prepared in S2 in a gold plating apparatus to a thickness of 10 nm to 30 nm; Step S4, immersing the titanium dioxide sprayed with gold in S4 in a prostate cancer DNA aptamer solution for modification, thereby obtaining a DNA-modified titanium dioxide thin film sensor; The DNA aptamer probe sequences used to detect the target are as follows:

[0021] Specifically, S401, preparing a DNA solution; S402: Place the titanium dioxide sprayed with gold in S3 in the 1 nM / ml DNA prepared in S401 and modify it for 20 minutes.

[0022] like Figure 1 As shown in the figure, the micromorphology comparison of titanium dioxide nanotubes anodized at different voltages, AD is the SEM image of the front of the titanium dioxide nanotube, EH is the SEM image of the back of the titanium dioxide nanotube, different voltages will produce different electric field strengths, affecting the oxidation of titanium and the corrosion rate of fluoride ions, thereby affecting the size of the pores. After adjusting different parameters, the optimal voltage of 20V is set for oxidation.

[0023] like Figure 2 As shown in the figure, the morphology comparison of titanium dioxide nanotubes prepared under different anodizing time conditions is shown. In the figure, AE is the SEM image of the front of the titanium dioxide nanotube, and FJ is the SEM image of the back of the titanium dioxide nanotube. During the oxidation process at different times, there will be a balance between the formation of the oxide layer and the corrosion of fluoride ions. Too short a time will result in a thicker tube wall and a smaller diameter. Too long a time may cause the array to collapse and form a porous sponge shape. After adjusting different parameters, 1h is set as the optimal time for oxidation.

[0024] like Figure 3 The figure shows the change of ion transmission performance before and after gold sputtering. In the figure, A is the external voltage and external pressure ion current (IV), and B is the light energy ion current (It). On the one hand, surface gold plating will form a discontinuous island structure, which conducts electricity through the tunneling effect. On the other hand, it may produce a certain steric effect or introduce defects, thereby causing an increase in resistance and affecting ion transmission. After adjusting different parameters, a 10nm thick gold layer is set as the optimal condition.

[0025] like Figure 4 Figure 2 shows the cyclic stability of titanium dioxide nanotubes provided by the present invention. Titanium dioxide nanotubes prepared by anodization form a highly ordered vertically aligned structure, which effectively disperses stress during cyclic testing, reducing cracks or fractures caused by localized stress concentration. Furthermore, their hollow structure can buffer volume changes caused by repeated expansion and contraction of the material.

[0026] like Figure 5 As shown in the figure, it is a comparison of the contact angle changes before and after modification of the probe and target. The surface of the unmodified titanium dioxide nanotube is rich in hydroxyl groups, and the contact angle is usually hydrophilic. However, after gold plating, the polar groups on the surface are reduced, and the contact angle will increase significantly. The modification of DNA will introduce a phosphate backbone and base groups, which will increase the hydrophilicity.

[0027] Example 2 This embodiment provides an application of a titanium dioxide thin film sensor in detecting small biological molecules.

[0028] In step S5, the titanium dioxide thin film sensor prepared in Example 1 is used to perform current detection under conditions of external voltage and external light.

[0029] Specifically, S501: using a picoammeter to perform current detection using 1 μM / L potassium chloride as an electrolyte; S502: setting the picoammeter parameters to -1V to +1V under the external voltage condition, and the number of cycles is 5; S503: 365nm under external illumination, light intensity 29.3mW / cm 2 irradiation with ultraviolet light; S504: Use a picoammeter to detect current changes under the illumination of S503 light source Step S6, combining a blood sample containing 1 nM / ml of sarcosine with the aptamer and performing current detection under a voltage of -1 V to -1 V and external light illumination; Step S7, using the current changes detected in S5 and S6 for early diagnosis of prostate cancer.

[0030] Example 3 The only difference from Example 2 is that the concentration of the small molecule sarcosine in step S6 is 10 nM / ml, and the rest are the same.

[0031] Example 4 The only difference from Example 1 is that the concentration of the small molecule sarcosine in step S6 is 100 nM / ml, and the rest are the same.

[0032] Comparing the current detection results in Examples 2 to 4, the results are shown in the attached Figure 6 As shown, although the concentration of sarcosine is very low at 1 nM / L, the detected current change value has obvious fluctuations compared with higher concentrations, indicating that the detection limit of DNA modification for cancer small molecules is relatively high, and can reach a maximum concentration of 1 nM / ml.

[0033] Example 5 The only difference from Example 1 is that the intensity of the light in step S5 is 82.5 mW / cm 2 , and the rest are the same.

[0034] Example 6 The only difference from Example 1 is that the intensity of the light in step S5 is 121.5 mW / cm 2 , and the rest are the same.

[0035] Comparing the current detection results of Example 2, Example 5 and Example 6, as shown in the attached Figure 7 As shown, the light intensity is 121.5mW / cm 2 The 365nm UV photocurrent is the largest, which can be clearly compared with the PSA blood sample. This is because higher light intensity is more conducive to stimulating the separation of titanium dioxide electron holes, providing electron transition energy, thereby generating a larger photocurrent. Therefore, 121.5mW / cm 2 Optimal light intensity.

[0036] Example 7 The only difference from Example 1 is that only external voltage detection is adopted in step S5, and the rest are the same.

[0037] Compare Example 7 with Example 2, as shown in the attached Figure 8 From a scientific point of view, the electron current under voltage alone is smaller than the ion current with photoelectric effect, and lacks the ion current generated by electron-hole separation, so it works best under ultraviolet light.

[0038] Example 8 The difference from Example 1 is only that the concentration of potassium chloride is different, and all the other are the same.

[0039] like Figure 9As shown in the figure, the test current is basically proportional to the concentration. However, in biological monitoring, lower concentrations can significantly reduce the ion mobility in the electrolyte, thereby reducing the non-Faraday current and improving the signal-to-noise ratio. Therefore, the optimal concentration of the experiment is 1μM / L.

[0040] Example 9 The only difference from Example 1 is that different cancer macromolecular marker proteins are detected.

[0041] In biological detection, DNA modification generally reacts with target proteins by specific binding, such as Figure 10 As shown, the comparison of the detection results of different proteins IV and It showed that the modified DNA had a significant response to the cancer marker protein PSA.

[0042] Example 10 The only difference from Example 1 is that different small molecule cancer markers are detected.

[0043] In biological detection, DNA modification generally produces a response by specifically binding to target molecules, such as Figure 11 As shown, by comparing the IV and It detection results of different molecules, it was shown that the modified DNA in the experiment had a significant response to the cancer marker sarcosine.

[0044] Example 11 The only difference from Example 1 is that the test sample is a real blood sample from Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology.

[0045] like Figure 12 As shown in the figure, under the same other conditions, the specific response signal change results of real blood samples from patients and healthy people are detected by DNA. Compared with healthy people, patients have obvious current fluctuations and a greater signal change rate.

[0046] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a DNA-modified titanium dioxide thin film sensor, characterized in that: The following steps are involved: S1, anodizing the titanium sheet into a titanium dioxide nanotube array, A two-electrode system is used, with a titanium sheet as the working electrode and a platinum sheet as the auxiliary electrode. A voltage of 20-25 V is applied to the surface of the titanium sheet for 30-90 minutes to form a titanium dioxide nanotube array. S2. The prepared titanium dioxide nanotubes are sprayed with gold in a gold plating apparatus. S3. Immerse the gold-sprayed titanium dioxide in a thiol-modified DNA solution for modification, thereby obtaining the DNA-modified titanium dioxide thin film sensor; the DNA is an aptamer of the biomolecule to be detected.

2. The preparation method according to claim 1, wherein In step S2, the thickness of the gold spraying is 10-30 nm.

3. A titanium dioxide thin film sensor prepared by the preparation method according to claims 1-2.

4. Use of the titanium dioxide thin film sensor as claimed in claim 3 in detecting biomolecules.

5. A device for detecting biomolecules, characterized in that: The method comprises two chambers, wherein the titanium dioxide thin film sensor according to claim 3 is arranged between the two chambers, an ultraviolet light source is arranged above the titanium dioxide thin film sensor, and both ends of the titanium dioxide thin film sensor are connected to the two chambers respectively. The two chambers are filled with potassium chloride solution and are respectively provided with a working electrode and a reference electrode. The transmembrane ion current is recorded before and after the addition of the biological small molecule sample to be tested.

6. The device according to claim 5, characterized in that The wavelength of the ultraviolet light source is 364 nm, and the intensity range is 82.5-121.5 mW / cm 2 .

7. The device according to claim 6, characterized in that The concentration of the potassium chloride solution is not less than 1 μM / L.

8. A method for detecting markers for early diagnosis of prostate cancer, characterized in that: Using the device as claimed in claim 5, a picoammeter is used to measure the transmembrane ion current before and after the addition of the test sample, and the current change rate is calculated. The formula is: Current change rate % = (I - I0) / I0, Where I0 is the current value measured before the sample to be tested is added, and I is the current value measured after the sample to be tested is added.

9. The method according to claim 8, wherein The diagnostic marker is sarcosine or PSA protein.

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