Photo-thermal enhanced cascade gated nano-channel as well as preparation method and application thereof

Abstract

The invention discloses a photo-thermal enhanced cascade gated nanochannel as well as a preparation method and application thereof, and belongs to the technical field of detection. According to the photo-thermal enhanced cascade gating nano-channel, titanium nano-channels which are of a tubular structure and are parallel to each other serve as a base body, the two ends of each titanium nano-channel are in a large opening shape and a small opening shape, and gating structures with a cascade relation are formed at the small opening ends and the large opening ends respectively; growing gold nanoparticles in situ on the end face of the small-opening end, and bonding a cDNA / C reactive protein aptamer compound to form a primary gating structure; a polydopamine film is generated at the large-opening end, and the zinc (ruthenium) metal organic framework material is anchored on the surface of the large-opening end through the polydopamine-mediated adhesion effect, so that a secondary gating structure is formed. The photo-thermal enhanced cascade gated nanochannel is used as a C-reactive protein detection platform, and is combined with the ELC technology, so that the detection accuracy is high, the detection period can be shortened, the detection sensitivity can be improved, and the detection of trace C-reactive protein in complex body fluid (such as serum) can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and in particular relates to a photothermal enhanced cascade gated nanochannel and a preparation method and application thereof. Background Art

[0002] In vivo, biological ion channels serve as crucial tools for exchanging substances with the surrounding environment. Embedded within cell membranes, biological ion channels can open and close in response to stimuli, regulating ion permeation across the cell membrane. This is crucial for regulating various physiological functions in cellular processes. Therefore, inspired by biological ion channels, nanochannels with similar functions have been a hot research area, potentially facilitating the development of intelligent nanomachines for future applications, such as molecular filtration, nanofluidic devices, and biosensors. Nanochannels, with their controllable geometry, tunable surface properties, and excellent mechanical stability, have shown great potential for applications in molecular filters, biosensors, and energy conversion devices. Compared to traditional biosensors, sensors constructed with nanochannels offer significant advantages in bioanalytical detection, including high sensitivity, high specificity, and high spatiotemporal resolution, due to their unique nanoscale spatial effects. Nanochannels respond to various external stimuli, such as pH, temperature, voltage, ions, light, and target analytes. Designing asymmetric nanochannels with unique ion transport properties, such as ion selectivity, ion gating, and ion rectification, is of great significance. Asymmetric nanochannels exhibit distinct characteristics along their structure, such as high selectivity and gating effects. When functional gate switches are asymmetrically installed on a nanochannel, target recognition reactions can precisely open the gate, allowing molecules / ions to enter and achieving selective ion transport. However, nanochannels also have limitations in analyzing and detecting actual samples, such as high ion transport resistance, which leads to low transport efficiency.

[0003] In recent years, the photothermal enhancement effect (PTE) has attracted widespread attention as an effective, non-invasive, and low-toxic method for hyperthermia detection. Various photothermal agents have been extensively explored over the past few years, including gold nanoparticles, carbon nanomaterials, semiconductor materials, and organic polymers. Among them, gold nanoparticles (AuNPs) are considered one of the most promising nanobiomaterials for photothermal effects due to their strong near-infrared absorption, high photothermal conversion efficiency, excellent thermal conductivity, good cytocompatibility, and lack of significant in vivo toxicity. In recent years, the photothermal effect induced by near-infrared (NIR) laser irradiation of AuNPs has been used to control drug release and enhance reaction rates. The photothermal effect of AuNPs can significantly promote the dynamic diffusion of ions and molecules, enhance molecular thermal motion, promote reactant mass transfer, and accelerate chemical reaction kinetics. The localized surface plasmon resonance (LSPR) effect of gold nanoparticles enhances the efficient transfer of interfacial hot electrons, significantly improving the kinetics of photothermal reactions. In addition, some polymers, such as polydopamine (PDA), have conjugated systems and aromatic ring structures that can efficiently absorb near-infrared (NIR) light and convert it into heat energy. Local temperature increases can effectively accelerate the thermal motion of nearby molecules and increase the diffusion rate. Based on this, AuNPs and PDA with photothermal enhancement effects can be combined with nanochannels to overcome the limitations of slow mass transfer rates in nanochannels, thereby accelerating reaction speeds and improving detection efficiency. However, due to the interference of background signals, improving the sensitivity of biomarker detection in nanochannels still faces huge challenges.

[0004] Currently, the vast majority of nanochannel sensing platforms utilize transmembrane ionic current as the signal response for target analysis. However, the asymmetric migration of anions and cations at the conical nanochannel orifice leads to ion rectification, and the time-consuming nature of functionalized gated nanochannels in capturing trace amounts of target analytes in biological samples affects their sensitivity and selectivity to a certain extent, limiting the scope of application of this method. Electrochemiluminescence (ECL) is the excited-state light emission produced by free radicals in electron transfer reactions on the electrode surface. ECL combines the advantages of electrochemistry and chemiluminescence, with high sensitivity, low background signal, simple instrumentation, fast detection speed, and easy operation. Therefore, ECL is a powerful bioassay and clinical diagnostic analysis technology. Currently, the detection of ECL in complex body fluids still faces huge challenges. Summary of the Invention

[0005] In response to the problems existing in the prior art, the purpose of the present invention is to provide a photothermally enhanced cascade-gated nanochannel and a preparation method thereof, and to combine the thermally enhanced cascade-gated nanochannel with ELC technology for the detection of C-reactive protein in serum, thereby realizing low background signal, high efficiency, high sensitivity and simple operation of C-reactive protein detection in complex body fluids.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a photothermally enhanced cascade-gated nanochannel. The nanochannel is based on a tubular, parallel titanium nanochannel (TiNM) with large and small openings at each end. Cascade-like gating structures are formed at the small and large ends, respectively. Gold nanoparticles (AuNPs) are in situ grown on the end surface of the small end and bonded to a cDNA / C-reactive protein aptamer complex, forming a primary gating structure. A polydopamine film (PDA) is formed on the large end, and a zinc (ruthenium) metal organic framework (ZnMOF(Ru)) is anchored to the surface of the large end through polydopamine-mediated adhesion, forming a secondary gating structure. Both the gold nanoparticles and the polydopamine have a photothermal enhancement effect, ensuring uniform heating at both ends of the nanochannel.

[0008] Furthermore, in the cDNA / C-reactive protein aptamer complex, the cDNA sequence is: 5'-CCCCAGACACGG-SH-3', the thiol group on the cDNA forms an Au-S bond with the gold nanoparticles, and then is modified to the small end of the titanium nanochannel. The C-reactive protein aptamer is: 5'-CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC-3', the C-reactive protein aptamer and cDNA become DNA double strands through base complementary pairing, and then form a "closed gate" at the small end.

[0009] Zinc (ruthenium) metal organic framework materials with Zn 2+ As the metal node, tris(4,4-dicarboxybipyridyl)ruthenium chloride (Ru(dcbpy)3 2+ ) is the ligand, Ru(dcbpy)3 2+ It is a luminescent substance with ECL activity.

[0010] The present invention also provides a method for preparing a photothermally enhanced cascade-gated nanochannel, which specifically comprises the following steps:

[0011] (1) Preparation of titanium nanochannels: The titanium sheet is pretreated, then electrochemically anodized, and then immersed in a H2O2 solution for demolding to obtain titanium nanochannels with open ends. After natural drying, the sheet is annealed in air. Finally, the sheet is immersed in a TiCl4 solution, then cleaned and blown dry to obtain titanium nanochannels with large and small openings at both ends. TiCl4 is hydrolyzed to obtain TiO2, which can effectively reduce the functional pore size of the nanochannel, which is conducive to obtaining an ideal gating effect.

[0012] (2) Preparation of a primary gated titanium nanochannel: gold nanoparticles are modified at the small end of the titanium nanochannel by chemical reduction; a cDNA solution is simultaneously prepared in a buffer solution and incubated at room temperature, and the cDNA is activated; the small end of the titanium nanochannel modified with gold nanoparticles is then placed in a cDNA solution for cDNA modification, incubated at room temperature in the dark, and then washed with a buffer solution; the small end of the titanium nanochannel modified with cDNA and gold nanoparticles is then placed in a C-reactive protein aptamer solution, incubated at room temperature, and then thoroughly washed with a buffer solution to remove unbound aptamers, thereby obtaining a primary gated titanium nanochannel;

[0013] (3) Preparation of photothermally enhanced cascade-gated nanochannels: The large-mouth end of the primary gating structure titanium nanochannel is connected to a container, and a polydopamine solution is added to the container and stirred to form a polydopamine film at the large-mouth end, which is then washed with deionized water; a solution of zinc (ruthenium) metal organic framework material is simultaneously synthesized using a water bath heating method and stored at room temperature; the solution of zinc (ruthenium) metal organic framework material is evenly spin-coated on the polydopamine film, and the polydopamine film modified with zinc (ruthenium) metal organic framework material at the large-mouth end forms a secondary gating structure, which is then dried at room temperature to obtain a photothermally enhanced cascade-gated nanochannel.

[0014] Furthermore, the pretreatment of the titanium sheet in step (1) is to place the cut titanium sheet in isopropyl alcohol, ethanol and deionized water for ultrasonic cleaning to remove impurities on the surface of the titanium sheet, and then blow dry with inert gas;

[0015] Electrochemical anodization is to place the pretreated titanium sheet in an ethylene glycol / lactic acid electrolyte containing NH4F, with a platinum sheet as the cathode and the pretreated titanium sheet as the anode; the concentration of NH4F in the ethylene glycol / lactic acid electrolyte containing NH4F is 0.05mol / L-0.15mol / L, the voltage is 110V-140V, and the anodization time is 15min-25min.

[0016] Furthermore, the volume fraction of the H2O2 solution in the demoulding treatment in step (1) is 30%-35%;

[0017] The annealing temperature is 400-500°C, the heating rate is 2-4°C / min, and the time is 1.5-2.5 hours;

[0018] The concentration of TiCl4 solution is 0.05mol / L-0.15mol / L, the soaking temperature is 65℃-75℃, and the soaking time is 1.5h-2.5h.

[0019] Furthermore, step (2) modifies the gold nanoparticles at the small end of the titanium nanochannel by chemical reduction, which comprises spraying a HAuCl4 solution with a concentration of 0.4 mmol / L-0.6 mmol / L onto the surface of the small end of the titanium nanochannel, placing the solution in an independent container, and then placing the solution together with an independent container containing a NaBH4 / ethanol solution in a sealed container, performing steam reduction by heating, washing, and drying with an inert gas to obtain a titanium nanochannel with the small end modified with gold nanoparticles.

[0020] Preferably, the concentration of the NaBH4 / ethanol solution is 1.5 mmol / L, the temperature of the steam reduction is 70°C, and the time is 30 min.

[0021] Furthermore, the buffer for preparing the cDNA solution in step (2) is a HAc-NaAc buffer, and the cDNA is activated by adding 8 mmol / L-12 mmol / L of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to the HAc-NaAc buffer containing the cDNA solution, and the incubation time at room temperature is 0.5 h-1.5 h;

[0022] The small end of the titanium nanochannel modified with gold nanoparticles is placed in a cDNA solution for cDNA modification, incubated in the dark at room temperature for 10 h to 14 h, and washed with a Tris-HCl buffer;

[0023] The small end of the titanium nanochannel modified with cDNA and gold nanoparticles was placed in a C-reactive protein aptamer solution and incubated at room temperature for 2 hours. The buffer used for washing was Tris-HCl buffer.

[0024] Furthermore, the stirring time of adding the polydopamine solution in step (3) is ≥10 min to form a polydopamine film on the surface of the large end of the nanochannel;

[0025] A water bath heating method is used to synthesize a zinc (ruthenium) metal organic framework material solution, comprising dissolving Zn(NO3)2·6H2O, polyvinylpyrrolidone (PVP), Ru(dcbpy)3Cl2, and pyrazine in H2O, followed by ultrasonic treatment to obtain a uniform solution, heating the solution in a water bath to 75°C-85°C, reacting the solution for 10 hours-14 hours, washing the obtained crystals with water by centrifugation, drying the solution at 55°C-65°C, and then dispersing the solution in water. The obtained zinc (ruthenium) metal organic framework material solution is stored at room temperature for later use.

[0026] The solution of zinc (ruthenium) metal organic framework material is spin-coated on the polydopamine film at a rotation speed of 300 rpm-600 rpm and a spin-coating time of 10 s-60 s. The obtained photothermal enhanced cascade gated nanochannel is stored at 4° C. for future use.

[0027] The present invention also provides an application of a photothermal enhanced cascade-gated nanochannel, combined with ELC technology, for the detection of C-reactive protein in serum.

[0028] Furthermore, an electrochemical sensing platform for detecting C-reactive protein in serum was constructed based on the photothermal-enhanced cascade-gated nanochannel, which also included an electrolytic cell consisting of a sample cell and a detection cell. The two ends of the photothermal-enhanced cascade-gated nanochannel were clamped between the sample cell and the detection cell by transparent silicone sheets, with the small end facing the sample cell and the large end facing the detection cell. A three-electrode system was used for testing, with a platinum wire as a counter electrode in the sample cell, a gold electrode as a working electrode in the detection cell, and Ag / AgCl as a reference electrode. 50 mmol / L Tris-HCl buffer solution with a pH of 7.4 was added to the sample cell and the detection cell, respectively. 0.1 mmol / L adenosine triphosphate (ATP) and a C-reactive protein serum solution of the to-be-tested concentration were added to the sample cell, and a quartz window was set on the small end side of the sample cell so that a near-infrared laser was irradiated through the quartz window onto the small end surface of the photothermal-enhanced cascade-gated nanochannel.

[0029] The testing process is as follows:

[0030] Before the ECL test begins, the C-reactive protein serum solution to be tested is incubated in the sample cell at room temperature, and then the small end of the photothermally enhanced cascade-gated nanochannel is irradiated with a near-infrared laser. Then, 50 mmol / L of K2S2O8 is added to the detection cell for ECL testing.

[0031] The principle of the photothermal-enhanced cascade-gated nanochannel used in the detection platform of C-reactive protein in serum is as follows: the small and large ends of the cascade-gated nanochannel are modified with AuNPs and polydopamine with photothermal effects, respectively. By irradiating with near-infrared light, the temperature of the nanochannel and the surrounding area is increased, and the molecular thermal motion on the surface of the nanomembrane and in the nanochannel is accelerated. On the one hand, the specific binding speed of C-reactive protein in the serum to be tested and the C-reactive protein aptamer modified at the small end can be accelerated, and the primary gate can be quickly opened. On the other hand, after passing through the primary gate, ATP smoothly enters the nanochannel and quickly diffuses to the secondary gate under the action of photothermal effect, capturing the ECL luminescent material Ru(dcbpy)3 in the ZnMOF(Ru) 2+ , releasing it into the detection pool to generate ECL signal with the co-reaction reagent K2S2O8, thereby realizing the detection of the target C-reactive protein.

[0032] Advantages and effects of the present invention:

[0033] 1. High sensitivity and low background signal, combined with electrochemiluminescence (ECL) technology, using the luminescent material (Ru(dcbpy) released by ZnMOF(Ru) 3+) generates signals with the characteristics of "low background interference and high signal-to-noise ratio", which is particularly suitable for the detection of trace biomarkers (C-reactive protein) in complex body fluids (such as serum).

[0034] 2. Photothermal enhancement improves detection efficiency. Gold nanoparticles (AuNPs) and polydopamine (PDA) are modified at both ends of the nanochannel, respectively. The two generate a photothermal effect under the irradiation of near-infrared light (808nm), significantly accelerating the molecular thermal motion: 1) increasing the binding speed of C-reactive protein and aptamer (first-level gating quickly opens); 2) promoting the diffusion rate of ATP in the channel and accelerating the release of ECL luminophore by the second-level gating (ZnMOF(Ru)).

[0035] 3. Cascade gating enhances selectivity with a dual-stage gating design: 1) Primary gating (small-mouth end): Target screening is achieved through specific recognition of C-reactive protein by the aptamer; 2) Secondary gating (large-mouth end): ATP captures the luminophore in the ZnMOF (Ru) and triggers the ECL signal; the cascade mechanism reduces nonspecific adsorption and improves detection accuracy.

[0036] 4. Synergistic effects optimize performance. The localized surface plasmon resonance (LSPR) effect of AuNPs enhances the local electric field, improves electron transfer efficiency, and further strengthens the ECL response. PDA acts as an adhesion layer to improve the stability of ZnMOF (Ru) and prevent luminescent material leakage.

[0037] 5. The preparation process is controllable, and the pore size of the nanochannel is regulated by TiCl4 hydrolysis (the small end is reduced to ~20nm), optimizing the gating effect; the modification positions of AuNPs and ZnMOF (Ru) are precise (small end / large end), ensuring clear functional zoning.

[0038] 6. The dual gating mechanism of the photothermally enhanced cascade-gated nanochannel can achieve enhanced selectivity and signal transduction, which can be used to construct an electrochemiluminescence sensing platform that can achieve rapid and highly sensitive detection of C-reactive protein in complex body fluids.

[0039] 7. Based on the photothermal effect of AuNPs and PDA at both ends of the nanochannel, the temperature of the nanochannel and the surrounding area increases by irradiation with near-infrared light, the molecular thermal motion on the nanomembrane surface and in the nanochannel accelerates, the binding speed of C-reactive protein and aptamer accelerates, and the diffusion speed of ATP in the channel accelerates, thereby increasing the amount of ATP reacting with ZnMOF (Ru) per unit time and releasing Ru(dcbpy)3 2+ Therefore, under auxiliary lighting, the detection cycle can be shortened and the detection sensitivity can be improved.

[0040] 8. The C-reactive protein detection platform constructed in the present invention, assisted by 808nm near-infrared light, can enhance the local electric field and improve the electron transfer efficiency under the synergistic effect of the LSPR effect of AuNPs, ultimately improving the ECL response. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the preparation method of the photothermally enhanced cascade-gated nanochannel in Example 1;

[0042] Figure 2 Schematic diagram of the electrochemical sensing platform for detecting C-reactive protein in serum using photothermal-enhanced cascade-gated nanochannels in Example 1;

[0043] Figure 3 Scanning electron microscope (SEM) images of the TiNM obtained after annealing treatment in Example 1, wherein: (a) is the small end, (b) is the large end, (c) is the side view, and (d) is the enlarged side view;

[0044] Figure 4 The scanning electron microscope (SEM) images of the small-mouth end of the TiNM after immersion in TiCl4 solution in Example 1, where: (a) shows the TiO2 accumulation in the small-mouth end pore, and (b) shows the AuNPs distribution on the end surface of the small-mouth end;

[0045] Figure 5 This is a transmission electron microscopy (TEM) image of the small-mouth end of AuNPs / TiNM prepared in Example 1;

[0046] Figure 6 Characterization diagrams of the ZnMOF(Ru) material prepared in Example 1, wherein: (a) is a scanning electron microscope image of ZnMOF(Ru), (b) is a transmission electron microscope (TEM) image of ZnMOF(Ru), and (c) is a scanning electron microscope image of the ZnMOF(Ru) nanoflower structure;

[0047] Figure 7 The scanning electron microscope (SEM) images of the large-mouth end and small-mouth end of the gated nanochannel after the large-mouth end was surface-modified with ZnMOF (Ru) in Example 1, wherein: (a) is the large-mouth end of the gated nanochannel, and (b) is the small-mouth end of the gated nanochannel;

[0048] Figure 8 This is an atomic force microscope (AFM) image of the small end of the TiNM prepared in Example 1;

[0049] Figure 9 This is an atomic force microscope (AFM) image of the small-mouth end of AuNPs / TiNM prepared in Example 1;

[0050] Figure 10Atomic force microscopy (AFM) image of the small mouth end of Apt / AuNPs / TiNM prepared in Example 1. DETAILED DESCRIPTION

[0051] The present invention is described in detail below with reference to the embodiments.

[0052] The cDNA sequence used in each embodiment of the present invention is: 5'-CCCCAGACACGG-SH-3' (Shanghai Biotech), the C-reactive protein aptamer is: 5'-CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC-3' (Shanghai Biotech), and the deionized water used is secondary deionized water (>18 MΩ·cm).

[0053] Example 1

[0054] The present invention provides a method for preparing a photothermal enhanced cascade gated nanochannel, such as Figure 1 As shown, the specific steps include:

[0055] (1) Preparation of titanium nanochannels:

[0056] Pre-treatment of titanium sheets: Cut titanium sheets (16 mm × 16 mm × 0.1 mm) were placed in isopropyl alcohol (twice), ethanol (twice), and deionized water (once) and ultrasonicated for 30 min to remove surface impurities, and then dried with nitrogen.

[0057] A platinum sheet was used as cathode and a treated titanium sheet as anode, and the electrodes were placed in an ethylene glycol / lactic acid electrolyte containing 0.1 mol / L NH4F, with a volume ratio of ethylene glycol to lactic acid of 9:1, and anodic oxidation was performed at 120 V for 20 min.

[0058] After being taken out, the nanostructured TiNM was immersed in a 30% H2O2 solution to undergo a demolding treatment, obtaining a TiNM with two open ends.

[0059] After natural drying, the prepared TiNM was annealed in air at 450 °C for 2 h (heating rate 3 °C / min);

[0060] The annealed TiNM was placed in a 0.1 mol / L TiCl4 solution at 70°C and soaked for 2 h, washed three times with deionized water, blown dry with N2, and set aside.

[0061] (2) Preparation of primary gated titanium nanochannels:

[0062] 1 mL of 0.5 mmol / L HAuCl4 was sprayed onto the surface of the small-mouthed TiNM prepared in step (1) in five separate batches. The mixture was placed in a separate beaker and then placed together with the separate beaker containing the NaBH4 / ethanol solution in a large sealed beaker. The mixture was reacted at 70°C for 30 min and steam reduced to obtain AuNPs / TiNM with small-mouthed AuNPs. The mixture was washed three times with deionized water and then dried with N2.

[0063] 10 μL of 0.1 mol / L HAc-NaAc buffer solution (pH 5.0) and 10 μL of 10 mmol / L TCEP were added to 200 μL of thiol-modified cDNA (capture DNA, 2 μmol / L) solution and incubated at room temperature for 1 h. Then, AuNPs / TiNM was added and incubated at room temperature in the dark for 12 h. The AuNPs / TiNM modified with cDNA was placed in 2 μmol / L C-reactive protein aptamer. After incubation at room temperature for 2 h, the cDNA and C-reactive protein aptamer complex (Apt) was thoroughly washed with Tris-HCl (pH 7.4) buffer to remove unbound aptamer, resulting in a primary gated titanium nanochannel (Apt / AuNPs / TiNM) and stored at 4°C for future use.

[0064] (3) Preparation of photothermally enhanced cascade-gated nanochannels:

[0065] ZnMOF(Ru) was synthesized by water bath heating method. First, 10 mg Zn(NO3)2·6H2O, 20 mg PVP, 15 mg Ru(dcbpy)3Cl2, and 1.5 mg pyrazine were dissolved in 50 mL H2O in a 100 mL beaker. Subsequently, the solution was ultrasonically treated for 10 min to obtain a uniform solution. The beaker was then heated to 80 °C in a water bath and reacted for 12 h. The obtained orange crystalline sample was washed with water by centrifugation several times and dried at 60 °C. It was then dispersed in water (1.5 mg / mL) and stored at room temperature for later use. The luminophore Ru(dcbpy)3 with ECL activity in ZnMOF(Ru) was 2+ As a ligand, avoiding Ru(dcbpy)3 2+ To solve the leakage problem, we improved Ru(dcbpy)3 2+ utilization efficiency;

[0066] Apt / AuNPs / TiNM was placed in the middle of a homemade H-type diffusion cell, and 0.1 mg / mL dopamine solution (Tris-HCl buffer, pH 8.5) was added to the compartment on the inlet side of the large end and stirred for 10 minutes to form a PDA film at the large end. After the obtained membrane was gently washed with deionized water, a ZnMOF(Ru) solution was spin-coated on the PDA-modified side at a speed of 600 rpm for 15 seconds using a spin coater to prepare a uniform Apt / AuNPs / TiNM / ZnMOF(Ru), i.e., a photothermally enhanced cascade-gated nanochannel. The cell was dried at room temperature and stored at 4°C for future use. The mussel-inspired PDA can improve the interfacial compatibility between the surface layer and the support layer in the composite film. In the present invention, PDA can adhere to the nanochannel surface and the ZnMOF(Ru) as an interlayer to improve stability.

[0067] The photothermal enhanced cascade gated nanochannel prepared in Example 1 was used to detect C-reactive protein in serum. An electrochemical sensing platform for detecting C-reactive protein in serum was constructed based on the photothermal enhanced cascade gated nanochannel, and an H-type electrolytic cell consisting of a sample cell and a detection cell was prepared. Figure 2 As shown, the photothermally enhanced cascade-gated nanochannel is sandwiched between two transparent silicone sheets, which are then placed between two electrolytic cells, with the small end of the nanochannel facing the sample cell and the large end facing the detection cell. A three-electrode system is used for testing. In the sample cell, a platinum wire (0.5 mm in diameter, 30 mm in length) is used as the counter electrode. In the detection cell, a gold electrode (2 mm in diameter) is used as the working electrode, and Ag / AgCl is used as the reference electrode. The sample cell contains 0.1 mmol / L ATP and 50 mmol / L Tris-HCl buffer solution (pH 7.4) of different concentrations of C-reactive protein to be tested. The detection cell contains 50 mmol / L Tris-HCl buffer solution (pH 7.4), a quartz window is set on the left end face of the sample cell. Near-infrared laser (808nm) passes through the quartz window and irradiates the small end surface of the photothermal enhanced cascade gated nanochannel. Based on the photothermal enhancement effect of AuNPs and PDA, the local temperature rises, accelerating the thermal motion of nearby molecules, further accelerating the binding speed of C-reactive protein and the aptamer gate in the sample cell and the diffusion rate of ATP, realizing the rapid detection of biomolecules. When the target analyte C-reactive protein is present, the specific binding of C-reactive protein and the aptamer opens the primary gate "gate", allowing ATP to smoothly enter the nanochannel and reach the secondary gate "gate". ATP combines with ZnMOF (Ru) to release the ECL luminescent material Ru(dcbpy)3 2+ It enters the detection pool and reacts with the co-reaction reagent K2S2O8 to generate ECL signal, thereby realizing the detection of the target C-reactive protein.

[0068] Testing process:

[0069] Before the ECL test began, the C-reactive protein solution of the serum to be tested in the sample pool was incubated at room temperature for 30 minutes, and then the small mouth end was irradiated with a near-infrared laser (808 nm) for 30 minutes. Subsequently, 50 mmol / L K2S2O8 was added to the detection pool for ECL testing. The scanning potential range of the ECL test was -1.6 to 0 V, and the photomultiplier tube voltage was 700 V.

[0070] Performance Analysis:

[0071] (1) Preparation and characterization:

[0072] The TiNM obtained after annealing in step (1) of the preparation method of Example 1 was characterized by SEM. Figure 3 As shown in (a) and 3(b), the TiNM prepared by electrochemical anodization is composed of many dense conical asymmetric nanochannels, which are open at both ends. The pore size of the small end is about 45±5nm, and the pore size of the large end is about 100±10nm. Figure 3 As shown in (c), the thickness of the prepared TiNM is about 38 ± 0.5 μm. Figure 3 As shown in (d), TiNMs are tubular and parallel to each other.

[0073] The small end of the TiNM after being immersed in the TiCl4 solution in step (1) of the preparation method of Example 1 was characterized by SEM. Figure 4 As shown in (a), it can be seen that the small TiO2 particles formed by hydrolysis are tightly packed in the small end pores, and the effective pore size is reduced to 20nm; Figure 4 As shown in (b), AuNPs are evenly distributed on the end surface of the nanochannel.

[0074] The AuNPs / TiNM small-mouth end prepared in step (2) of the preparation method of Example 1 was characterized by TEM. Figure 5 As shown, AuNPs are aggregated in a rod-like shape, and the size of these nanoparticles is about 7 nm. The interplanar spacing of TiNM is 0.248 nm, corresponding to the (004) crystal plane of anatase (JPCDS No. 21-1272), indicating that AuNPs are successfully modified onto the small end of TiNM.

[0075] The ZnMOF (Ru) material prepared in step (3) of the preparation method of Example 1 was characterized by SEM and TEM. Figure 6 (a) Figure 6 As shown in (b), it can be seen that ZnMOF(Ru) presents a “nanoflower structure”, which is composed of thin nanosheets, such as Figure 6 As shown in (c), the thickness of the nanosheets is about 50 nm, indicating that ZnMOF(Ru) was successfully prepared.

[0076] The large-mouth end of the TiNM of Example 1 was modified. After the surface of the large-mouth end was modified with ZnMOF (Ru) material, the large-mouth end and the small-mouth end of the gated nanochannel were characterized by SEM. Figure 7 As shown in (a), it can be seen that the ZnMOF (Ru) nanosheets are evenly distributed on the surface of the large end of the nanochannel; Figure 7 As shown in (b), the channel structure on the surface of the small end is clearly visible, and no ZnMOF (Ru) nanosheets are modified, which shows that the modification method of the present invention can prevent the solution from penetrating into the small end during the modification of the large end; it further shows that the present invention successfully modified the small end of the nanochannel with AuNPs and the large end with ZnMOF (Ru) through the asymmetric modification method. In the present invention, the modifications at the large and small ends of the channel show obvious anisotropy.

[0077] The surface morphology of the small-mouth end of TiNM, AuNPs / TiNM, and Apt / AuNPs / TiNM prepared in Example 1 was analyzed by atomic force microscopy. Figure 8 As shown in Figure 2, the unmodified TiNM exhibits a highly ordered and uniformly distributed pore structure; Figure 9 As shown in Figure 2, after chemical reduction of AuNPs, small particles were obviously attached to the surface of TiNM; Figure 10 As shown in Figure 3, after assembly with the cDNA / C-reactive protein aptamer, the surface of the small end of the TiNM becomes rougher and the end becomes irregular in shape, indicating that the sample is coated with a layer of biomacromolecules.

[0078] Example 2

[0079] The method for preparing a photothermally enhanced cascade-gated nanochannel of the present invention specifically comprises the following steps:

[0080] (1) Preparation of titanium nanochannels:

[0081] Pre-treatment of titanium sheets: Cut titanium sheets (16 mm × 16 mm × 0.1 mm) were placed in isopropyl alcohol (twice), ethanol (twice), and deionized water (once) and ultrasonicated for 30 min to remove surface impurities, and then dried with nitrogen.

[0082] A platinum sheet was used as cathode and a treated titanium sheet as anode, and the electrodes were placed in an ethylene glycol / lactic acid electrolyte containing 0.05 mol / L NH4F, with a volume ratio of ethylene glycol to lactic acid of 9:1, and anodic oxidation was performed at 110 V for 60 min.

[0083] After being taken out, the nanostructured TiNM was immersed in a 35% H2O2 solution to undergo a demolding treatment, obtaining a TiNM with two open ends.

[0084] After natural drying, the prepared TiNM was annealed in air at 500 °C for 3 h (heating rate 4 °C / min);

[0085] The annealed TiNM was placed in a 0.05 mol / L TiCl4 solution at 75°C and soaked for 1.5 h, washed three times with deionized water, blown dry with N2, and set aside.

[0086] (2) Preparation of primary gated titanium nanochannels:

[0087] 1 mL of 0.4 mmol / L HAuCl4 was sprayed onto the surface of the small-mouthed TiNM prepared in step (1) in five separate batches. The mixture was placed in a separate beaker and then placed together with the separate beaker containing the NaBH4 / ethanol solution in a large sealed beaker. The mixture was reacted at 70°C for 30 min and steam reduced to obtain AuNPs / TiNM with small-mouthed AuNPs. The mixture was washed three times with deionized water and then dried with N2.

[0088] 10 μL of 0.1 mol / L HAc-NaAc buffer solution (pH 5.0) and 10 μL of 8 mmol / L TCEP were added to 200 μL of thiol-modified cDNA (capture DNA, 2 μmol / L) solution and incubated at room temperature for 1 h. Then, AuNPs / TiNM was added and incubated at room temperature in the dark for 14 h. The AuNPs / TiNM modified with cDNA was placed in 2 μmol / L C-reactive protein aptamer. After incubation at room temperature for 2 h, the cDNA and C-reactive protein aptamer complex (Apt) was thoroughly washed with Tris-HCl (pH 7.4) buffer to remove unbound aptamer, resulting in the primary gated structure titanium nanochannel (Apt / AuNPs / TiNM) and stored at 4°C for future use.

[0089] (3) Preparation of photothermally enhanced cascade-gated nanochannels:

[0090] ZnMOF(Ru) was synthesized by water bath heating method. First, 10 mg Zn(NO3)2·6H2O, 20 mg PVP, 15 mg Ru(dcbpy)3Cl2, and 1.5 mg pyrazine were dissolved in 50 mL H2O in a 100 mL beaker. Subsequently, the solution was ultrasonicated for 10 min to obtain a uniform solution. The beaker was then heated to 75 °C in a water bath and reacted for 14 h. The obtained orange crystalline sample was washed with water by centrifugation several times and dried at 60 °C. It was then dispersed in water (1.5 mg / mL) and stored at room temperature for later use. The ECL-active luminophore Ru(dcbpy)3 in ZnMOF(Ru) was 2+ As a ligand, avoiding Ru(dcbpy)32+ To solve the leakage problem, we improved Ru(dcbpy)3 2+ utilization efficiency;

[0091] Apt / AuNPs / TiNM was placed in the middle of a homemade H-type diffusion cell, and 0.1 mg / mL dopamine solution (Tris-HCl buffer, pH 8.5) was added to the compartment on the inlet side of the large end and stirred for 10 minutes to form a PDA film at the large end. After the obtained film was gently washed with deionized water, ZnMOF(Ru) solution was spin-coated on the PDA-modified side at a speed of 300 rpm for 60 seconds using a spin coater to prepare a uniform Apt / AuNPs / TiNM / ZnMOF(Ru), i.e., a photothermally enhanced cascade-gated nanochannel. The resulting film was dried at room temperature and stored at 4°C for future use.

[0092] Example 3

[0093] The method for preparing a photothermally enhanced cascade-gated nanochannel of the present invention specifically comprises the following steps:

[0094] (1) Preparation of titanium nanochannels:

[0095] Pre-treatment of titanium sheets: Cut titanium sheets (16 mm × 16 mm × 0.1 mm) were placed in isopropyl alcohol (twice), ethanol (twice), and deionized water (once) and ultrasonicated for 30 min to remove surface impurities, and then dried with nitrogen.

[0096] A platinum sheet was used as cathode and a treated titanium sheet as anode, and the electrodes were placed in an ethylene glycol / lactic acid electrolyte containing 0.15 mol / L NH4F, with a volume ratio of ethylene glycol to lactic acid of 9:1, and anodic oxidation was performed at 140 V for 10 min.

[0097] After being taken out, the nanostructured TiNM was immersed in a 32% H2O2 solution to undergo a demolding treatment, obtaining a TiNM with two open ends.

[0098] After natural drying, the prepared TiNM was annealed in air at 550 °C for 1.5 h (heating rate 2 °C / min);

[0099] The annealed TiNM was placed in a 0.15 mol / L TiCl4 solution at 65°C and soaked for 2.5 h, washed three times with deionized water, dried with N2, and set aside.

[0100] (2) Preparation of primary gated titanium nanochannels:

[0101] 1 mL of 0.6 mmol / L HAuCl4 was sprayed onto the surface of the small-mouthed TiNM prepared in step (1) in five separate batches. The mixture was placed in a separate beaker and then placed together with the separate beaker containing the NaBH4 / ethanol solution in a large sealed beaker. The mixture was reacted at 70°C for 30 min and steam reduced to obtain AuNPs / TiNM with small-mouthed AuNPs. The mixture was washed three times with deionized water and then dried with N2.

[0102] 10 μL of 0.1 mol / L HAc-NaAc buffer solution (pH 5.0) and 10 μL of 12 mmol / L TCEP were added to 200 μL of thiol-modified cDNA (capture DNA, 2 μmol / L) solution and incubated at room temperature for 1 h. Then, AuNPs / TiNM was added and incubated at room temperature in the dark for 10 h. The AuNPs / TiNM modified with cDNA was placed in 2 μmol / L C-reactive protein aptamer. After incubation at room temperature for 2 h, the cDNA and C-reactive protein aptamer complex (Apt) was thoroughly washed with Tris-HCl (pH 7.4) buffer to remove unbound aptamer, resulting in the primary gated structure titanium nanochannel (Apt / AuNPs / TiNM) and stored at 4°C for future use.

[0103] (3) Preparation of photothermally enhanced cascade-gated nanochannels:

[0104] ZnMOF(Ru) was synthesized by water bath heating method. First, 10 mg Zn(NO3)2·6H2O, 20 mg PVP, 15 mg Ru(dcbpy)3Cl2, and 1.5 mg pyrazine were dissolved in 50 mL H2O in a 100 mL beaker. Subsequently, the solution was ultrasonicated for 10 min to obtain a uniform solution. The beaker was then heated to 85°C in a water bath and reacted for 10 h. The obtained orange crystalline sample was washed with water by centrifugation several times and dried at 60°C. It was then dispersed in water (1.5 mg / mL) and stored at room temperature for later use. The luminophore Ru(dcbpy)3 with ECL activity in ZnMOF(Ru) was 2+ As a ligand, avoiding Ru(dcbpy)3 2+ To solve the leakage problem, we improved Ru(dcbpy)3 2+ utilization efficiency;

[0105] Apt / AuNPs / TiNM was placed in the middle of a homemade H-type diffusion cell, and 0.1 mg / mL dopamine solution (Tris-HCl buffer, pH 8.5) was added to the compartment on the inlet side of the large end and stirred for 10 minutes to form a PDA film at the large end. After the obtained film was gently washed with deionized water, ZnMOF(Ru) solution was spin-coated on the PDA-modified side at a speed of 600 rpm for 10 seconds using a spin coater to prepare a uniform Apt / AuNPs / TiNM / ZnMOF(Ru), i.e., a photothermally enhanced cascade-gated nanochannel. The resulting film was dried at room temperature and stored at 4°C for later use.

Claims

1. A photothermally enhanced cascade-gated nanochannel, characterized in that: The matrix is ​​a tubular titanium nanochannel with parallel openings at both ends, with large and small openings, forming cascade-like gating structures at the small and large ends respectively; gold nanoparticles are in situ grown on the end face of the small end and bonded to a cDNA / C-reactive protein aptamer complex to form a primary gating structure; a polydopamine film is generated at the large end, and the zinc (ruthenium) metal organic framework material is anchored to the surface of the large end through polydopamine-mediated adhesion to form a secondary gating structure.

2. The photothermally enhanced cascade-gated nanochannel according to claim 1, wherein: In the cDNA / C-reactive protein aptamer complex, the cDNA sequence is: 5'-CCCCAGACACGG-SH-3', and the C-reactive protein aptamer is: 5'-CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC-3'. The C-reactive protein aptamer and cDNA form a DNA double strand through base complementary pairing; Zinc (ruthenium) metal organic framework materials with Zn 2+ It is the metal node and tris(4,4-dicarboxybipyridyl)ruthenium chloride is the ligand.

3. A method for preparing the photothermally enhanced cascade-gated nanochannel according to claim 1, characterized in that: The specific steps include: (1) Preparation of titanium nanochannels: The titanium sheet is pretreated, then electrochemically anodized, and then immersed in a H2O2 solution for demolding to obtain titanium nanochannels with open ends. After natural drying, the sheet is annealed in air. Finally, the sheet is immersed in a TiCl4 solution, then washed and dried to obtain titanium nanochannels with large and small openings at both ends. (2) Preparation of a primary gated titanium nanochannel: gold nanoparticles are modified at the small end of the titanium nanochannel by chemical reduction; a cDNA solution is simultaneously prepared in a buffer solution and incubated at room temperature, and the cDNA is activated; the small end of the titanium nanochannel modified with gold nanoparticles is then placed in a cDNA solution for cDNA modification, incubated at room temperature in the dark, and then washed with a buffer solution; the small end of the titanium nanochannel modified with cDNA and gold nanoparticles is then placed in a C-reactive protein aptamer solution, incubated at room temperature, and then thoroughly washed with a buffer solution to obtain a primary gated titanium nanochannel; (3) Preparation of photothermally enhanced cascade-gated nanochannels: The large-mouth end of the primary gating structure titanium nanochannel is connected to a container, and a polydopamine solution is added to the container and stirred to form a polydopamine film at the large-mouth end, which is then washed with deionized water; a solution of zinc (ruthenium) metal organic framework material is simultaneously synthesized using a water bath heating method and stored at room temperature; the solution of zinc (ruthenium) metal organic framework material is evenly spin-coated on the polydopamine film, and the polydopamine film modified with zinc (ruthenium) metal organic framework material at the large-mouth end forms a secondary gating structure, which is then dried at room temperature to obtain a photothermally enhanced cascade-gated nanochannel.

4. The method for preparing a photothermally enhanced cascade-gated nanochannel according to claim 3, wherein: The pretreatment of the titanium sheet in step (1) is to place the cut titanium sheet in isopropyl alcohol, ethanol and deionized water for ultrasonic cleaning to remove impurities on the surface of the titanium sheet, and then blow dry with inert gas; Electrochemical anodization is to place the pretreated titanium sheet in an ethylene glycol / lactic acid electrolyte containing NH4F, with a platinum sheet as the cathode and the pretreated titanium sheet as the anode; the concentration of NH4F in the ethylene glycol / lactic acid electrolyte containing NH4F is 0.05mol / L-0.15mol / L, the voltage is 110V-140V, and the anodization time is 15min-25min.

5. The method for preparing a photothermally enhanced cascade-gated nanochannel according to claim 3, wherein: The volume fraction of the H2O2 solution in the demoulding treatment in step (1) is 30%-35%; The annealing temperature is 400-500°C, the heating rate is 2-4°C / min, and the time is 1.5-2.5 hours; The concentration of TiCl4 solution is 0.05mol / L-0.15mol / L, the soaking temperature is 65℃-75℃, and the soaking time is 1.5h-2.5h.

6. The method for preparing a photothermally enhanced cascade-gated nanochannel according to claim 3, wherein: In step (2), gold nanoparticles are modified at the small end of the titanium nanochannel by a chemical reduction method, wherein a HAuCl4 solution with a concentration of 0.4 mmol / L-0.6 mmol / L is sprayed onto the surface of the small end of the titanium nanochannel and placed in an independent container. The solution is then placed together with an independent container containing a NaBH4 / ethanol solution in a sealed container, and subjected to steam reduction, washing, and drying with an inert gas to obtain a titanium nanochannel with gold nanoparticles modified at the small end.

7. The method for preparing a photothermally enhanced cascade-gated nanochannel according to claim 3, wherein: The buffer for preparing the cDNA solution in step (2) is HAc-NaAc buffer, and the cDNA is activated by adding 8 mmol / L-12 mmol / L of tris(2-carboxyethyl)phosphine hydrochloride to the HAc-NaAc buffer containing the cDNA solution, and the incubation time at room temperature is 0.5 h-1.5 h; The small end of the titanium nanochannel modified with gold nanoparticles is placed in a cDNA solution for cDNA modification, incubated in the dark at room temperature for 10 h to 14 h, and washed with a Tris-HCl buffer; The small end of the titanium nanochannel modified with cDNA and gold nanoparticles was placed in a C-reactive protein aptamer solution and incubated at room temperature for 2 hours. The buffer used for washing was Tris-HCl buffer.

8. The method for preparing a photothermally enhanced cascade-gated nanochannel according to claim 3, wherein: The stirring time of adding the polydopamine solution in step (3) is ≥10 min; A solution of a zinc (ruthenium) metal organic framework material is synthesized by a water bath heating method, comprising dissolving Zn(NO3)2·6H2O, polyvinyl pyrrolidone, Ru(dcbpy)3Cl2, and pyrazine in H2O, followed by ultrasonic treatment to obtain a uniform solution, heating the solution in a water bath to 75°C-85°C, reacting the solution for 10 hours-14 hours, washing the obtained crystals with water by centrifugation, drying the solution at 55°C-65°C, and then dispersing the solution in water. The obtained solution of the zinc (ruthenium) metal organic framework material is stored at room temperature for future use. The solution of zinc (ruthenium) metal organic framework material is spin-coated on the polydopamine film at a rotation speed of 300 rpm-600 rpm and a spin-coating time of 10 s-60 s. The obtained photothermal enhanced cascade gated nanochannel is stored at 4° C. for future use.

9. An application of the photothermally enhanced cascade-gated nanochannel according to claim 1, characterized in that: Combined with ELC technology, it is used to detect C-reactive protein in serum.

10. The use of the photothermally enhanced cascade-gated nanochannel according to claim 9, characterized in that: An electrochemical sensing platform for detecting C-reactive protein in serum was constructed based on a photothermal-enhanced cascade-gated nanochannel, which also included an electrolytic cell consisting of a sample cell and a detection cell. The two ends of the photothermal-enhanced cascade-gated nanochannel were clamped between the sample cell and the detection cell by transparent silicone sheets, with the small end facing the sample cell and the large end facing the detection cell. A three-electrode system was used for testing, with a platinum wire as a counter electrode in the sample cell, a gold electrode as a working electrode in the detection cell, and Ag / AgCl as a reference electrode. 50 mmol / L Tris-HCl buffer solution with a pH of 7.4 was added to the sample cell and the detection cell, respectively. 0.1 mmol / L adenosine triphosphate and a C-reactive protein serum solution to be tested were added to the sample cell, and a quartz window was set on the small end of the sample cell to allow a near-infrared laser to irradiate the small end surface of the photothermal-enhanced cascade-gated nanochannel through the quartz window. The testing process is as follows: Before the ECL test begins, the C-reactive protein serum solution to be tested is incubated in the sample cell at room temperature, and then the small end of the photothermally enhanced cascade-gated nanochannel is irradiated with a near-infrared laser. Then, 50 mmol / L of K2S2O8 is added to the detection cell for ECL testing.

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