Polydopamine modified high-entropy alloy nanosheet as well as preparation method and application thereof

Abstract

The invention discloses a polydopamine modified high-entropy alloy nanosheet as well as a preparation method and application thereof, and belongs to the technical field of metal functional material preparation. A salt template method is adopted, various metal precursors and a sodium chloride template are mixed, high-temperature annealing is conducted, the template is removed, and the ultrathin and porous five-element high-entropy alloy nanosheet is synthesized. Then, through in-situ auto-polymerization of dopamine under an alkaline condition, a uniform polydopamine functional layer is formed on the surface of the nanosheet; the composite material has high conductivity and high catalytic activity of the high-entropy alloy nanosheet and excellent biocompatibility and rich surface functional groups of the polydopamine. The composite material is further used as a sensing interface to fix and identify an aptamer of ProGRP, and an electrochemical aptamer sensor for ultrasensitive detection of a small cell lung cancer marker ProGRP is constructed. The sensor has the advantages of wide detection range, high sensitivity, good selectivity and strong stability, and has a wide application prospect in the fields of early diagnosis of cancers and precise medical treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of metal functional material preparation, in particular to a kind of polydopamine modified high-entropy alloy nanosheet and its preparation method and application. BACKGROUND

[0002] Small cell lung cancer (SCLC) is a subtype of lung cancer with high malignancy and poor prognosis. ProGRP (pro-gastrin-releasing peptide) has been recognized as a highly specific serological marker for the diagnosis and monitoring of SCLC. Currently, the clinical detection of ProGRP mainly relies on enzyme-linked immunosorbent assay (ELISA) and other methods, but these methods have the problems of complicated operation, long time-consuming, limited sensitivity and high cost.

[0003] Electrochemical aptamer sensors combine the high specificity and high affinity of aptamers with the rapid and sensitive advantages of electrochemical detection, and are strong competitors for the next generation of disease diagnosis tools. However, its performance largely depends on the properties of the sensing interface material. An ideal interface material should have a high specific surface area to immobilize a large number of recognition molecules, excellent electrical conductivity to facilitate electron transfer, good biocompatibility to maintain biological activity, and abundant functional groups to achieve stable biological fixation.

[0004] Currently, it is a technical problem to be solved in the art to construct an electrochemical aptamer sensor for detecting ProGRP with high sensitivity and strong stability. SUMMARY

[0005] Therefore, the present application aims to overcome the shortcomings of the prior art and provide a polydopamine modified high-entropy alloy nanosheet and its preparation method, and then a high-performance ProGRP electrochemical aptamer sensor based on the material.

[0006] High-entropy alloys (HEA) are solid solutions formed by five or more main metal elements in equal atomic ratio or near equal atomic ratio, which exhibit excellent catalytic activity and stability due to their unique "cocktail effect", lattice distortion effect and slow diffusion effect. Preparing them into two-dimensional nanosheets (HEANSs) can further expose a large number of active sites. However, the surface of HEANSs is inert, making it difficult to directly and efficiently immobilize biological molecules. Polydopamine (PDA) is a bio-polymer inspired by mussel adhesive proteins, which can form a firm adhesive coating on the surface of various materials and introduce a large number of active groups such as amino groups and phenolic hydroxyl groups, providing an ideal platform for the immobilization of biological molecules. Currently, there is no report on the use of PDA functionalized HEANSs composite materials to construct electrochemical aptamer sensors for detecting ProGRP.

[0007] To achieve the above object, in a first aspect, the application provides a polydopamine modified high-entropy alloy nanosheet, which is composed of a high-entropy alloy nanosheet core and a polydopamine shell layer coated on the surface of the core; the high-entropy alloy nanosheet contains five metal elements of iron, cobalt, copper, nickel and iridium.

[0008] The molar ratio of the five metal elements in the high-entropy alloy nanosheet is 1-1.5: 1-1.5: 1-1.5: 1-1.5: 1-1.5.

[0009] In a second aspect, the application provides a preparation method of the polydopamine modified high-entropy alloy nanosheet. S1, preparation of high-entropy alloy nanosheet: dissolving five metal precursors of Fe(acac)2, Co(acac)2, Cu(acac)2, Ni(acac)2 and Ir(acac)2 in ethanol, adding sodium chloride powder, stirring and mixing, grinding the mixture into fine powder after drying, annealing the fine powder in an inert atmosphere, washing to remove the sodium chloride template after cooling, and vacuum drying to obtain ultra-thin and porous high-entropy alloy nanosheet; S2, polydopamine functionalization: dispersing the high-entropy alloy nanosheet obtained in step S1 in Tris-HCl buffer solution, adding dopamine hydrochloride, stirring and reacting, allowing the dopamine to polymerize on the surface of the nanosheet, centrifuging, washing and vacuum drying after the reaction is completed, to obtain the polydopamine modified high-entropy alloy nanosheet, denoted as HEANSs@PDA.

[0010] Preferably, in step S1, the molar ratio of the five metal precursors is 1-1.5: 1-1.5: 1-1.5: 1-1.5: 1-1.5; the volume-to-total-substance amount ratio of the ethanol to the five precursors is 60-80 mL: 2.5-3 mmol; the mass-to-total-substance amount ratio of the sodium chloride powder to the five precursors is 40-60 g: 2.5-3 mmol; the stirring and mixing time is 48 h; the drying temperature of the mixture is 60℃; the high-temperature annealing temperature is 700-900℃, and the time is 1-4 h; and the vacuum drying temperature is 60℃.

[0011] Preferably, in step S2, the pH of the Tris-HCl buffer solution is 8.0-9.0; the mass-to-volume ratio of the high-entropy alloy nanosheet to the Tris-HCl buffer solution is 10 mg: 50-100 mL; the volume ratio of the high-entropy alloy nanosheet to dopamine hydrochloride is 1: 2-3; the stirring and reaction time is 8-16 h; and the vacuum drying temperature is 60℃.

[0012] In a third aspect, the present application provides an electrochemical aptamer sensor, wherein a working electrode of the electrochemical aptamer sensor is prepared by drop coating a polydopamine modified high-entropy alloy nanosheet suspension on a glassy carbon electrode surface, air-drying, and then fixing a ProGRP specific nucleic acid aptamer.

[0013] In a fourth aspect, the present application provides a use of the above-mentioned electrochemical aptamer sensor in the preparation of a diagnostic reagent or device for detecting a small cell lung cancer marker, i.e., a gastrin releasing peptide precursor.

[0014] The electrochemical aptamer sensor has a linear range of 100 pg / mL to 10 μg / mL and a detection limit of less than 1 pg / mL.

[0015] Compared with the prior art, the present application provides a polydopamine modified high-entropy alloy nanosheet, a preparation method and application thereof, which have the following beneficial effects: 1) The five-membered HEANSs synthesized by a salt template method are combined with PDA for the first time to create a new type of nanocomposite, realizing deep synergy of structure and function. The core of HEANSs has excellent intrinsic conductivity and rich catalytic sites due to the lattice distortion caused by the high-entropy effect, but it is difficult to biologically functionalize the metal surface. The PDA shell firmly covers the HEANSs through strong coordination, and the rich functional groups such as amino and phenolic hydroxyl groups provide a stable covalent fixation platform for aptamers and enhance the dispersibility and biocompatibility of the material. The combination of the two creates a "high-efficiency electron transport core" and a "stable biological interface shell", making the composite material have high sensitivity and high specificity in electrochemical sensing, which is the key to achieving ultra-low detection limit and excellent stability.

[0016] 2) The salt template method is simple, low-cost, easy to scale up, and can accurately control the morphology and composition of the nanosheet. The PDA functionalization process is mild and universal, without damaging the intrinsic properties of HEANSs.

[0017] 3) The aptamer sensor based on the material exhibits an extremely wide detection range (10 μg / mL -100 pg / mL), an ultra-low detection limit (0.874 pg / mL), excellent repeatability (RSD=1.78%) and long-term stability (the signal remains 96.248% after 9 days). In actual serum sample detection, the recovery rate is high, which proves its great potential for clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments. Obviously, the drawings in the following description only constitute a part of the embodiments of the present application, and for those skilled in the art, other drawings can be obtained based on the provided drawings without creative labor.

[0019] Figure 1 Figure 1 is a characterization diagram of HEANSs@PDA composite material. Wherein (A) is a SEM diagram, (B) is a TEM diagram, (C-D) are HR-TEM-EDS element distribution diagrams.

[0020] Figure 2 Figure 4 is a DPV response curve of different concentrations of ProGRP.

[0021] Figure 3 Figure 5 is a calibration curve. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments only constitute a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0023] Embodiment 1: Preparation of HEANSs@PDA composite material S1, accurately weigh 0.6 mmol of Fe(acac)2, Co(acac)2, Cu(acac)2, Ni(acac)2 and Ir(acac)2 respectively, dissolve them in 60 mL of anhydrous ethanol together, and magnetically stir for 30 minutes; add 60 g of NaCl powder to the solution, and stir vigorously at room temperature for 48 hours, then dry the mixture in an oven at 60℃, and then grind it into fine powder with a mortar; place the fine powder in a tube furnace, and under nitrogen atmosphere, increase the temperature to 800℃ at a rate of 5℃ / min, and keep for 2 hours; after natural cooling to room temperature, centrifugally wash the product with a large amount of deionized water until no white precipitate is detected in the supernatant with AgNO3 solution, indicating that the NaCl template has been completely removed, and then dry it in vacuum at 60℃ to obtain high-entropy alloy nanosheets (HEANSs).

[0024] S2, take 10 mg of the above HEANSs, disperse in 50 mL of Tris-HCl buffer solution (10 mM) with pH=8.5, ultrasonic for 30 minutes to make it fully dispersed; add 20 mg of dopamine hydrochloride to it, slowly stir at room temperature for 12 hours; after the end, centrifugal collection of black solid, and washed with deionized water and ethanol three times alternately, finally 60 ℃ vacuum drying, to obtain the polydopamine modified high-entropy alloy nanosheet, recorded as HEANSs@PDA.

[0025] Example 2: Preparation of HEANSs@PDA composite material S1, accurately weigh Fe(acac)2 0.6 mmol, Co(acac)2 0.5 mmol, Cu(acac)2 0.4 mmol, Ni(acac)2 0.4 mmol and Ir(acac)2 0.6 mmol, dissolve in 80 mL of anhydrous ethanol together, magnetic stirring for 30 minutes; add 40 g of NaCl powder to the solution, stir vigorously at room temperature for 48 hours, then dry the mixture in an oven at 60 ℃, followed by grinding into fine powder with a mortar; place the fine powder in a tube furnace, under nitrogen atmosphere, increase the temperature to 800 ℃ at a rate of 5 ℃ / min, and keep for 2 hours; after natural cooling to room temperature, centrifugal wash the product with a large amount of deionized water until the supernatant is detected with AgNO3 solution without white precipitate, indicating that the NaCl template has been completely removed, and dried at 60 ℃ under vacuum to obtain high-entropy alloy nanosheet (HEANSs).

[0026] S2, take 10 mg of the above HEANSs, disperse in 100 mL of Tris-HCl buffer solution (10 mM) with pH=8.5, ultrasonic for 40 minutes to make it fully dispersed; add 30 mg of dopamine hydrochloride to it, slowly stir at room temperature for 12 hours; after the end, centrifugal collection of black solid, and washed with deionized water and ethanol three times alternately, finally 60 ℃ vacuum drying, to obtain the polydopamine modified high-entropy alloy nanosheet, recorded as HEANSs@PDA.

[0027] The prepared HEANSs@PDA was characterized, and the results are shown in Figure 1 From the figure, it can be seen that HEANSs@PDA has five kinds of metal elements, and has a sheet structure, proving to be a high-entropy alloy nanosheet.

[0028] Example 3: Construction of electrochemical aptamer sensor (1) The glassy carbon electrode with a diameter of 3 mm was polished with 0.3, 0.5 and 1.0 μm of alumina polishing powder on a suede in turn to mirror surface, and then ultrasonic cleaned in anhydrous ethanol and deionized water for 5 minutes, and dried with nitrogen.

[0029] (2) 8 μL of HEANSs@PDA aqueous dispersion solution with a concentration of 1 mg / mL was dropped on the surface of a clean glassy carbon electrode and dried at room temperature to obtain HEANSs@PDA / GCE.

[0030] (3) 9 μL of NH2-modified ProGRP aptamer (sequence: 5'-NH2-GGTTGGTGTGGTTGG-3') solution with a concentration of 3.5 μM was added dropwise to the surface of the modified electrode and incubated at room temperature for 35 minutes, followed by gentle washing with PBS buffer (0.1 M, pH 7.4) to remove the physically adsorbed aptamer, to obtain Apt / HEANSs@PDA / GCE.

[0031] (4) The prepared aptamer sensor was stored in a 4°C refrigerator for standby use.

[0032] Example 4: Detection performance of ProGRP The constructed Apt / HEANSs@PDA / GCE was incubated with a solution containing different concentrations of ProGRP (100 pg / mL to 10 μg / mL) at room temperature for 40 minutes. Subsequently, differential pulse voltammetry (DPV) test was performed in a solution containing 5 mM [Fe(CN)6] 3- / 4- and 0.1 M KCl, and the results are shown in Figure 2 . As can be seen from the graph, the DPV peak current significantly decreases with the increase of the concentration of ProGRP. The change in peak current was plotted against the logarithm of the concentration of ProGRP, and the results are shown in Figure 3 . As can be seen from the graph, a good linear relationship is shown in the range of 100 pg / mL to 10 μg / mL, and the detection limit is calculated to be 0.874 pg / mL (S / N=3). The sensor has no obvious response to other common cancer markers (such as CEA, PSA, AFP, etc.), showing high specificity.

[0033] The above description of the embodiments is for the purpose of enabling and using the invention for those of ordinary skill in the art. Those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A polydopamine-modified high-entropy alloy nanosheet, characterized in that, The polydopamine modified high-entropy alloy nanosheet is composed of a high-entropy alloy nanosheet core and a polydopamine shell coated on the surface of the high-entropy alloy nanosheet; the high-entropy alloy nanosheet contains five metal elements of iron, cobalt, copper, nickel and iridium.

2. The polydopamine-modified high-entropy alloy nanoplatelets according to claim 1, wherein, The molar ratio of the five metal elements in the high-entropy alloy nanosheet is 1-1.5: 1-1.5: 1-1.5: 1-1.5: 1-1.

5.

3. A method for preparing polydopamine-modified high-entropy alloy nanosheets according to any one of claims 1-2, characterized in that, Specifically comprising the following steps: S1, preparation of high-entropy alloy nanosheet: dissolving Fe(acac)2, Co(acac)2, Cu(acac)2, Ni(acac)2 and Ir(acac)2 five metal precursors in ethanol, adding sodium chloride powder, stirring and mixing, grinding the mixture into fine powder after drying, high-temperature annealing in inert atmosphere, washing to remove sodium chloride template after cooling, vacuum drying to obtain ultra-thin and porous high-entropy alloy nanosheet; S2, polydopamine functionalization: dispersing the high-entropy alloy nanosheet obtained in step S1 in Tris-HCl buffer solution, adding hydrochloric acid dopamine, stirring and reacting to make dopamine polymerize on the surface of the nanosheet, centrifuging, washing and vacuum drying after the reaction is completed to obtain the polydopamine modified high-entropy alloy nanosheet, denoted as HEANSs@PDA.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the five metal precursors in step S1 is 1-1.5: 1-1.5: 1-1.5: 1-1.5: 1-1.5; The volume to total amount of substance ratio of the five precursors of the ethanol is 60-80 mL: 2.5-3 mmol; The mass to total amount of substance ratio of the five precursors of the sodium chloride powder is 40-60 g: 2.5-3 mmol; The stirring and mixing time is 48 h; The drying temperature of the mixture is 60℃; The high-temperature annealing temperature is 700-900℃, and the time is 1-4 h; The vacuum drying temperature is 60℃.

5. The preparation method according to claim 3, characterized in that, The pH of the Tris-HCl buffer solution in step S2 is 8.0-9.0; The volume to mass ratio of the Tris-HCl buffer solution to the high-entropy alloy nanosheet is 50-100 mL: 10 mg; The volume ratio of the high-entropy alloy nanosheet to hydrochloric acid dopamine is 1: 2-3; The stirring reaction time is 8-16 h; The vacuum drying temperature is 60℃.

6. An electrochemical aptamer sensor, characterized in that, The working electrode of the electrochemical aptamer sensor is prepared by dropping coating the polydopamine modified high-entropy alloy nanosheet suspension of any one of claims 1-2 on the surface of a glassy carbon electrode, air-drying, and then fixing the ProGRP specific nucleic acid aptamer.

7. Use of the electrochemical aptamer sensor of claim 6 in the preparation of a diagnostic reagent or device for detecting the small cell lung cancer marker gastrin-releasing peptide precursor.

8. Use according to claim 7, characterized in that, The linear range of the electrochemical aptamer sensor for detection is 100 pg / mL to 10 μg / mL, and the detection limit is lower than 1 pg / mL.

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