Preparation method and application of TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor

Abstract

The invention provides a preparation method of a TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor and an application of the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor. Comprising the following steps: step 1, preparing TiO2 / FTO; step 2, preparation of TiO2 / Au-C3N4 (titanium dioxide / Au-C3N4); step 3, preparing a nucleic acid aptamer; and step 4, using the confining liquid mercaptohexanol. The preparation method comprises the following steps: firstly, preparing a TiO2 thin film by an electrophoretic deposition method, and then combining the TiO2 thin film with graphite phase carbon nitride (g-C3N4) doped with high-conductivity noble metal gold nanoparticles (Au NPs) to form a heterojunction; under an illumination condition, Au NPs and photons interact to generate a regional surface plasma resonance phenomenon, and a strong electromagnetic field and high-concentration high-energy carriers are formed on the surface of the nano structure in the process, so that the photocurrent response of the composite material is remarkably enhanced; besides, the introduced Au NPs can also form an Au-S bond with the DNA aptamer containing the-SH group, and more binding sites are provided, so that the capture capability of the aptamer is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a preparation method and application of a TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor. Background Art

[0002] Alzheimer's disease (AD) is widely regarded as one of the most serious global health crises in the 21st century, and its high incidence and risk of disease have attracted widespread attention. The disease not only significantly reduces the quality of life of the elderly, but also brings heavy economic and care burdens to families and societies in an aging society, becoming a major challenge facing the medical community and society. Studies have shown that early diagnosis plays a vital role in the treatment of AD and improving patient prognosis. However, due to the extremely low content of target proteins in real clinical specimens of AD patients (such as cerebrospinal fluid, plasma, saliva, etc.), complex components and individual differences, and the existing early diagnosis methods including positron emission tomography (PET), enzyme-linked immunosorbent assay (ELISA), protein immunoblotting (Western Blot), single molecule microarray (SiMoA), electrochemiluminescence immunoassay (ECLI), etc., which are usually invasive, costly and have limited sensitivity, these methods are not suitable for large-scale application.

[0003] Therefore, the detection and application of AD biomarkers in clinical specimens currently faces severe challenges. To address this problem, the market urgently needs to develop a simple, rapid, non-invasive, low-cost and highly sensitive accurate detection technology to help clinicians achieve early diagnosis of AD patients. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of a TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor.

[0005] The present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention relates to a method for preparing a TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor, comprising the following steps:

[0007] Step 1, Preparation of TiO2 / FTO

[0008] (1.1) Weigh titanium dioxide and iodine and add them to acetone to prepare an electrophoresis suspension;

[0009] (1.2) The suspension was sonicated in an ultrasonic machine for 30 minutes and stirred for 1.5 hours until the powder was evenly dispersed;

[0010] (1.3) Use two cleaned FTO glasses as anode and cathode respectively to perform electrophoretic deposition operation;

[0011] (1.4) After the electrophoresis is completed, the FTO glass deposited with the TiO2 film is annealed at 400°C for 120 minutes to obtain a TiO2 / FTO sensor;

[0012] Step 2, Preparation of TiO2 / Au-C3N4

[0013] (2.1) Mix melamine and phosphorous acid, add water and mix well, and stir in a water bath at 80°C for 1 hour;

[0014] (2.2) Add 20 μL of 10 g / mL chloroauric acid solution to the solution in (2.1) above and stir evenly, then heat and filter, rinse repeatedly to remove phosphorous acid, and dry to obtain a powder;

[0015] (2.3) Take the powder obtained in (2.2), mix it with glycerol and ethanol, reflux at 110°C for 3 h, filter and dry the obtained sample, heat it to 600°C in a muffle furnace at 2°C / min, anneal it for 2 h, and obtain Au-C3N4;

[0016] (2.4) Add appropriate amount of ethanol and water to the obtained Au-C3N4, ultrasonicate for 6-8h, and let stand until the solution is clear; dry at 80℃, calcine at elevated temperature, and anneal for 30min to obtain TiO2 / Au-C3N4 sensor;

[0017] Step 3, preparation of nucleic acid aptamers

[0018] Place 1.0 nmol of nucleic acid aptamer solution in a centrifuge and centrifuge at 6000 rpm for 5 minutes. Add 1 ml of PBS buffer to dilute the sample and mix thoroughly. Take 10 μL of the dilution and evenly apply it on the surface of the TiO2 / Au-C3N4 sensor. Place it in an incubator at 37°C and incubate it for 90 minutes.

[0019] Step 4, use of blocking liquid mercaptohexanol

[0020] 10 microliters of 1.0 mM MCH was drop-coated on the surface of the TiO2 / Au-C3N4 / aptamer sensor and incubated in a 37°C incubator for 1 hour to block the redundant nonspecific binding sites on the sensor surface. The blocking molecule formed on the aptamer and used to block the redundant sites on the aptamer and the functionalized molecular connection layer is mercaptohexanol.

[0021] Preferably, in step 1, the ratio of the titanium dioxide powder to the iodine element is 4:1.

[0022] Preferably, in step 1, the TiO2 in the TiO2 / FTO sensor is a base layer, which presents a dense and uniform spherical distribution of TiO2.

[0023] Preferably, in step 2, the usage ratio of melamine to phosphorous acid is 1:1.2.

[0024] Preferably, in step 2, the ratio of glycerol to ethanol is 1:3.

[0025] Preferably, in step 2, the temperature-raising calcination is to raise the temperature from 30° C. to 250° C. in a muffle furnace.

[0026] Preferably, in step 2, Au-C3N4 in the TiO2 / Au-C3N4 sensor is a functionalized molecular connecting layer formed on the TiO2 substrate layer.

[0027] Preferably, the functional molecular connection layer presents an irregular two-dimensional sheet structure.

[0028] Preferably, the functionalized molecular connection layer is used to provide a functional group site for connecting the nucleic acid aptamer, namely gold nanoparticles (Au-NPs), and connect the nucleic acid aptamer with -SH through the Au-S bond.

[0029] Preferably, in step 3, the nucleic acid aptamer is fixed on the functionalized molecular connection layer, wherein the sequence of the nucleic acid aptamer is 5'-SH-GCT GCC TGT GGT GTT GGG GCG GGT GCG-3', which corresponds to the Alzheimer's disease biomarker Aβ40.

[0030] The present invention adopts electrophoretic deposition technology to prepare TiO2 thin film on FTO glass substrate, which has the advantages of being green, environmentally friendly and low-cost. Although TiO2 has a wide band gap of 3.2eV, the surface charge transfer efficiency is low and it is easily affected by the recombination of electron-hole pairs, which limits the sensitivity and stability of TiO2 biosensors.

[0031] In a second aspect, the present invention also relates to the use of the aforementioned TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor for detecting the Alzheimer's disease biomarker Aβ40.

[0032] The present invention has the following advantages:

[0033] (1) The present invention adopts methods such as doping and compounding of narrow bandgap materials that match the energy level to form a heterojunction of TiO2 and g-C3N4, thereby effectively improving the performance of the sensor, promoting the effective separation and transfer of electron-hole pairs, and enhancing the photocurrent response.

[0034] (2) The present invention doped g-C3N4 with highly conductive precious metal gold (Au) nanoparticles (Au NPs). Under illumination conditions, Au NPs interact with photons to produce a localized surface plasmon resonance (LSPR) phenomenon. During this process, a strong electromagnetic field and a high concentration of high-energy carriers are formed on the surface of the nanostructure, which significantly enhances the photocurrent response of the composite material. In addition, the introduced Au NPs can also form Au-S bonds with DNA aptamers containing -SH groups, providing more binding sites, thereby enhancing the capture ability of the aptamer.

[0035] (3) The sensor of the present invention can be used for quantitative detection of amyloid Aβ40 protein in AD patients in different body fluid environments, and has extremely high sensitivity, good biocompatibility and excellent detection performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The electrode morphology of the present invention under the conditions of 35V, 45V and 55V electrophoretic deposition voltage under SEM scanning electron microscope;

[0037] Figure 2 TEM transmission electron microscope image of the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor prepared by the present invention;

[0038] Figure 3 The element distribution diagram of the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor prepared by the present invention;

[0039] Figure 4 This is the XPS distribution diagram of the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor prepared by the present invention;

[0040] Figure 5 The photoelectric chemical biosensor prepared by the present invention is a detection performance diagram of the target protein molecule Aβ40 in the cerebrospinal fluid environment and the plasma environment;

[0041] Figure 6 A comparison chart of the selectivity of the photoelectrochemical biosensor prepared by the present invention for the target protein molecule Aβ40;

[0042] Figure 7 It is a demonstration diagram of the preparation of TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to the present invention. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0044] Example

[0045] This embodiment relates to a method for preparing a TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor. Figure 7 As shown, the following steps are included:

[0046] Step 1, Preparation of TiO2 / FTO

[0047] Weigh 0.048 g titanium dioxide and 0.012 g iodine and add them to acetone to prepare an electrophoresis suspension;

[0048] The suspension was sonicated in an ultrasonic machine for 30 min and then stirred on a mixing table for 1.5 h to ensure that the powder was evenly dispersed;

[0049] Two cleaned FTO (fluorine-doped tin oxide) glasses were used as anode and cathode respectively for electrophoretic deposition operation;

[0050] After the electrophoresis, the FTO glass with the TiO2 film deposited was annealed at 400°C for 120 minutes to improve the crystallinity and adhesion of the film, thereby obtaining a TiO2 / FTO sensor.

[0051] Step 2, Preparation of TiO2 / Au-C3N4

[0052] ① Mix 4g melamine and 4.8g phosphorous acid, add water and mix well, and stir in a water bath at 80℃ for 1 hour;

[0053] ② Add 20 μL of 10 g / mL chloroauric acid solution to the above solution and stir evenly, then heat and filter, rinse repeatedly to remove phosphorous acid, and dry to obtain powder;

[0054] ③ Take all the powders in ②, mix with glycerol and ethanol (3:1), reflux at 110℃ for 3h (asbestos mesh temperature), filter and dry the obtained sample, heat to 600℃ in a muffle furnace at 2℃ / min, and anneal for 2h;

[0055] ④ Add appropriate amount of ethanol and water to the obtained Au-C3N4, ultrasonicate for 6-8h, and let stand until the solution is clear. The effective area of ​​the material is 0.5cm 2 , 900 μl of Au-C3N4 was drop-coated on each piece of material, dried on a drying table at 80 °C, calcined in a muffle furnace from 30 °C to 250 °C, and annealed for 30 min;

[0056] Step 3, Preparation and Use of Aptamers

[0057] Place 1.0 nmol of nucleic acid aptamer solution in a centrifuge and centrifuge at 6000 rpm for 5 minutes. Then, add 1 ml of PBS buffer to dilute the sample and mix thoroughly; take 10 μL of the dilution and evenly drop it on the prepared TiO2 / Au-C3N4 sensor surface; place the sensor in an incubator at 37°C for 90 minutes.

[0058] Step 4, use of blocking liquid mercaptohexanol

[0059] Take 10 μl of 1.0 mM MCH and drop it on the surface of the TiO2 / Au-C3N4 / nucleic acid aptamer sensor, incubate it in a 37°C incubator for 1 hour to block the excess nonspecific binding sites on the sensor surface, and finally obtain the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor.

[0060] The performance of the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor prepared in this example was tested.

[0061] Figure 1 It can be seen that when the deposition voltage is 35V, the TiO2 particles on the electrode surface are larger in size and the surface is relatively loose. This is because under a lower electric field, the movement speed of the positively charged TiO2 particles is slower, resulting in a looser film. When the deposition voltage is increased to 45V, the movement of the particles becomes more regular, and the TiO2 particles on the electrode surface become dense and uniform, which helps to prepare a more stable sensor. However, when the deposition voltage is further increased to 55V, the particles on the electrode surface have a loose structure again. This may be because the deposition rate of TiO2 particles is accelerated under higher voltages, making it difficult to form an ideal film state on the electrode surface within a limited time.

[0062] See Figure 2 As shown, Figure 2 (A) Dense and uniform titanium dioxide nanosphere structure can be observed, indicating that TiO2 can be successfully deposited on the FTO glass surface by EPD. Figure 2 (B) In the pure Au-C3N4 suspension, it can be clearly observed that the black granular substances are evenly distributed on the surface of the two-dimensional sheet-like g-C3N4, which are Au-NPs synthesized in the hydrothermal process. It is these Au-NPs that provide a large surface area and sufficient and firm active sites for the immobilization of biomolecules, thereby ensuring the effective transfer of electrons between the electrode and the electrolyte. Figure 2 (C) After the addition of Au-C3N4, irregular two-dimensional sheet structures are attached to the surface of titanium dioxide nanospheres, which is g-C3N4. The two structures are combined with each other to form an intimate heterojunction, which is conducive to the transport of carriers.

[0063] Figure 3 This is the TEM element mapping result of Au-C3N4 / TiO2 / FTO photoanode, in which Ti, O, C, N and Au elements are evenly distributed on the entire photoanode surface.

[0064] Figure 4 It can be seen that Figure 4 As shown in (A), the two peaks at 458.35 eV and 464.10 eV correspond to the characteristic peaks of Ti2 p3 / 2 and Ti2 p1 / 2 orbitals of TiO2, respectively, proving that Ti 4+ The existence of Figure 4 In (B), the characteristic peak at 529.55 eV is attributed to Ti-O, while the peak at 531.03 eV is attributed to -OH on the surface. Figure 4 As shown in (C), the three peaks located at 288.17 eV, 284.72 eV, and 286.12 eV belong to the sp2 orbitals of (CC) or (C=C), respectively. Figure 4 (D) The typical characteristic peaks of N 1s elements are 398.31 eV and 400.29 eV, corresponding to CN=C and CNH respectively. Figure 4 (E) The presence of Au element can be seen.

[0065] Figure 5 It can be seen that the linear relationship between the photocurrent density and the Aβ40 concentration of the sensor of the present invention for detecting Aβ40 in the cerebrospinal fluid environment is: Figure 5 In (A), Y = 1.406x + 2.631 (R 2 =0.99) The linear detection range of this sensor is 10 -15 ~10 -11 g / mL, the detection limit can reach 0.33fg / mL. In the plasma environment, the linear relationship between the photocurrent density and the Aβ40 concentration is: Figure 5 In (B), Y = 0.952x + 17.02 (R 2 =0.99) The linear detection range of this sensor is 10 -15 ~10 -11 g / mL, and the detection limit can reach 0.33fg / mL.

[0066] Figure 6 It can be seen that the selectivity of the sensor prepared by the present invention to Aβ40 was evaluated by simulating the complex environment of human body fluids and measuring the response of the sensor to Aβ42, tau, ascorbic acid, glucose, urea, and chitosan. The results showed that compared with Aβ40, the interfering substances with a concentration of 1000 times did not have a significant photocurrent response. Therefore, it is shown that the sensor has good selectivity for Aβ40.

[0067] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which will not affect the essence of the present invention.

Claims

1. A method for preparing a TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor, characterized in that: The following steps are involved: Step 1, Preparation of TiO2 / FTO (1.1) Weigh titanium dioxide and iodine and add them to acetone to prepare an electrophoresis suspension; (1.2) The suspension was sonicated in an ultrasonic machine for 30 minutes and stirred for 1.5 hours until the powder was evenly dispersed; (1.3) Use two cleaned FTO glasses as anode and cathode respectively to perform electrophoretic deposition operation; (1.4) After the electrophoresis is completed, the FTO glass deposited with the TiO2 film is annealed at 400°C for 120 minutes to obtain a TiO2 / FTO sensor; Step 2, Preparation of TiO2 / Au-C3N4 (2.1) Mix melamine and phosphorous acid, add water and mix well, and stir in a water bath at 80°C for 1 hour; (2.2) Add 20 μL of 10 g / mL chloroauric acid solution to the solution in (2.1) above and stir evenly, then heat and filter, rinse repeatedly to remove phosphorous acid, and dry to obtain a powder; (2.3) Take the powder obtained in (2.2), mix it with glycerol and ethanol, reflux at 110°C for 3 h, filter and dry the obtained sample, heat it to 600°C in a muffle furnace at 2°C / min, anneal it for 2 h, and obtain Au-C3N4; (2.4) Add appropriate amount of ethanol and water to the obtained Au-C3N4, ultrasonicate for 6-8h, and let stand until the solution is clear; dry at 80℃, calcine at elevated temperature, and anneal for 30min to obtain TiO2 / Au-C3N4 sensor; Step 3, preparation of nucleic acid aptamers Place 1.0 nmol of nucleic acid aptamer solution in a centrifuge and centrifuge at 6000 rpm for 5 minutes. Add 1 ml of PBS buffer to dilute the sample and mix thoroughly. Take 10 μL of the dilution and evenly apply it on the surface of the TiO2 / Au-C3N4 sensor. Place it in an incubator at 37°C and incubate it for 90 minutes. Step 4, use of blocking liquid mercaptohexanol Take 10 μl of 1.0 mM MCH and drop it on the surface of TiO2 / Au-C3N4 / nucleic acid aptamer sensor, and incubate it in a 37°C incubator for 1 hour to block the excess nonspecific binding sites on the sensor surface.

2. The method for preparing the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to claim 1, characterized in that: In step 1, the ratio of the titanium dioxide powder to the iodine element is 4:

1.

3. The method for preparing the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to claim 1, characterized in that: In step 1, TiO2 in the TiO2 / FTO sensor is a base layer, which presents a dense and uniform spherical distribution of TiO2.

4. The method for preparing the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to claim 1, characterized in that: In step 2, the usage ratio of melamine to phosphorous acid is 1:1.

2.

5. The method for preparing the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to claim 1, characterized in that: In step 2, the ratio of glycerol to ethanol is 1:

3.

6. The method for preparing the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to claim 1, characterized in that: In step 2, the temperature-raising calcination is to raise the temperature from 30° C. to 250° C. in a muffle furnace.

7. The method for preparing the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to claim 1, characterized in that: In step 2, Au-C3N4 in the TiO2 / Au-C3N4 sensor is a functionalized molecular connecting layer, which is formed on the TiO2 substrate layer.

8. The method for preparing the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to claim 7, characterized in that: The functional molecular connection layer presents an irregular two-dimensional sheet structure. The functionalized molecular connection layer is used to provide a functional group site for connecting the nucleic acid aptamer, namely gold nanoparticles (Au-NPs), and connect the nucleic acid aptamer with -SH through the Au-S bond.

9. The method for preparing the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor according to claim 1, characterized in that: In step 3, the nucleic acid aptamer is fixed on the functionalized molecular connection layer, wherein the sequence of the nucleic acid aptamer is 5'-SH-GCT GCC TGT GGT GTT GGG GCG GGT GCG-3', which corresponds to the Alzheimer's disease biomarker Aβ40.

10. An application of a TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor, characterized in that: Use of the TiO2 / Au-C3N4 heterojunction photoelectrochemical biosensor as claimed in claim 1 for detecting the Alzheimer's disease biomarker Aβ40.

Images