CN111060568A - Method for constructing collagen type III photoelectrochemical sensor based on rhenium disulfide nanosheet and application - Google Patents

Abstract

The invention discloses a method for constructing a triple-type collagen photoelectrochemical sensor based on a rhenium disulfide nanosheet and application thereof. According to the invention, the ionic liquid is used as a stripping agent and inserted or attached to the nano-sheet structure of the rhenium disulfide, so that the rhenium disulfide nano-sheet can be effectively prevented from agglomerating, the stripping efficiency is improved, the rhenium disulfide belongs to a direct band gap material, and the rhenium disulfide has better photoelectric property compared with other transition metal disulfides, and the high-selectivity and high-sensitivity analysis of the collagen type III is realized on the basis.

Description

Method for constructing collagen type III photoelectrochemical sensor based on rhenium disulfide nanosheet and application

Technical Field

The invention belongs to the technical field of novel nanometer functional materials and biosensing, and particularly relates to a method for constructing a collagen type III photoelectrochemical sensor based on a rhenium disulfide nanosheet and application of the collagen type III photoelectrochemical sensor.

Background

Abdominal Aortic Aneurysm (AAA) is a serious progressive degenerative disease, manifested by dilation of the abdominal aorta with degradation of the arterial wall extracellular matrix and development of chronic inflammation. AAA has a morbidity rate of 8% in older men over 65 years old, with men being slightly older than women and being the 13 th cause of death in our country. Smoking, advanced age and atherosclerosis have been identified as the most major risk factors for AAA induction. Most patients with AAA have no obvious clinical symptoms except occasional abdominal pain, and few patients have a tumor body which is already ruptured when the patients find the disease, the death rate after rupture is up to 80 percent, and the patients have serious harm to the health and life of the human bodies. Therefore, early screening and diagnosis are of great significance for preventing and treating abdominal aortic aneurysm or aortic dissection.

At present, ultrasonic examination, CT and MRI are the main means for screening abdominal aortic aneurysm or aortic dissection, but because of the limitation of medical conditions and cost, the large-scale early screening and prevention by the method is not convenient and fast. Studies have shown that abnormal expression of type III collagen plays an important role in the pathogenesis of abdominal aortic aneurysms. At present, methods for detecting the type III collagen mainly comprise a chromatography method, a mass spectrometry method and an enzyme-linked immunosorbent assay. The detection method has the defects of complex and tedious operation, low specificity and sensitivity, time consumption and the like. Therefore, it is important to provide a triple collagen sensor with low detection cost, high speed, high efficiency, high sensitivity and strong specificity.

The photoelectrochemical sensor has been paid attention to by more and more researchers in recent years due to the characteristics of high sensitivity, strong specificity, low detection cost and the like. The photoelectrochemical sensor is characterized in that when an external light source excites a photosensitive material to cause electron-hole pairs to be separated, and an electron donor occupies electron holes, electrons are transmitted on an electrode, a semiconductor, a modifier and an analyte to form photocurrent, the change of the concentration of the analyte can cause the change of the photocurrent, and the change can reflect the relationship between the concentration of the analyte and the photocurrent, so that the quantitative analysis of the analyte is realized. However, the technical difficulty of the photoelectrochemical sensor is how to increase the magnitude of the photocurrent and the stability thereof.

Transition metal disulfides are widely used in the fields of catalysis and photoelectricity due to their excellent photoelectrochemical properties. Rhenium disulfide (chemical formula is ReS)₂) The nano material is transition metal disulfide with direct band gap property, is a flaky two-dimensional layered structure nano material after being stripped, and has larger specific surface area and excellent photoelectrochemical activity. At present, the stripping method of the rhenium disulfide nanosheet mainly adopts water bath ultrasonic stripping in a single solvent, and the whole stripping process is long in time consumption and poor in stripping effect due to the fact that ultrasonic energy is not concentrated. Therefore, it is necessary to research a new method for peeling the rhenium disulfide nanosheet, so as to improve the peeling effect and shorten the peeling time.

Disclosure of Invention

The invention aims to provide a method for constructing a collagen type III photoelectrochemical sensor based on a rhenium disulfide nanosheet and application thereof, aiming at the problems in the prior art.

In order to achieve the purpose, the invention adopts the technical scheme that:

a preparation method of rhenium disulfide nanosheets based on ionic liquid assisted stripping comprises the following steps: dissolving 1-butyl-3-dodecyl imidazole bromide salt in N-methyl pyrrolidone, and adding ReS₂Subjecting the powder to ultrasonic treatment with a probe ultrasonic machine to obtain a dispersion of the ReS₂Centrifuging, washing and drying the solution of the nanosheets to obtain the ReS₂Nanosheets.

The invention also provides a preparation method of the 1-butyl-3-dodecyl imidazole bromide salt, which comprises the following steps:

(1) adding a small amount of sodium hydride into an acetonitrile solution containing imidazole for multiple times, reacting in ice bath to obtain an acetonitrile suspension of white imidazole sodium, and cooling to room temperature for later use;

(2) adding bromododecane into the suspension, stirring at reflux temperature overnight, performing suction filtration after the reaction is finished, taking the filtrate, and performing spin-drying on the solvent to obtain a crude product, and purifying to obtain a light yellow oily liquid dodecyl imidazole;

(3) and (3) dissolving the dodecyl imidazole and n-butyl bromide obtained in the step (2) in acetonitrile, stirring at the reflux temperature overnight, drying the solvent in a spinning mode after the reaction is finished to obtain a crude product, and purifying to obtain the yellow viscous oily liquid 1-butyl-3-dodecyl imidazole bromide salt.

As a further limitation of the above scheme, the molar ratio of sodium hydride, imidazole, bromododecane is 4:2: 1.

As a further limitation of the above scheme, in the step (3), the molar usage ratio of the dodecyl imidazole bromide salt to the n-butyl bromide is 1: 1-3.

As a further limitation of the above scheme, the 1-butyl-3-dodecyl imidazole bromide salt is reacted with ReS₂The dosage ratio of the powder is 2-8: 1.

As a further limitation of the scheme, the ultrasonic treatment time of the probe is 2-4 h, the power is 300W, and the ultrasonic machine is set to work for 5s and pause for 2 s.

As a further limitation of the above protocol, the centrifugation is performed for 5min at 500rpm and then for 15min at 12000 rpm. The invention firstly removes the block-shaped ReS by centrifuging for 5min at 500rpm₂Then will contain ReS₂Centrifuging the supernate of the nanosheets at 12000rpm to remove the solvent, thereby obtaining the ReS with excellent specific surface area and photoelectrochemical property₂Two-dimensional lamellar nanomaterials.

The invention also provides a method for constructing a collagen type III photoelectrochemical sensor based on the rhenium disulfide nanosheet, which comprises the following steps:

s1, pretreating a glassy carbon electrode: straight bare glassy carbon electrode (diameter 3mm) using 0.3 μm Al₂O₃Polishing the suspension, washing with distilled water, and sequentiallyAt HNO₃Ultrasonically cleaning the mixture by using ethanol and secondary distilled water, and airing the mixture for later use;

s2, taking 6-12 mu L of ReS prepared by any one of the methods₂The nano sheet dispersion liquid is dripped on the surface of the clean glassy carbon electrode obtained in the step S1 and dried at room temperature to prepare ReS₂a/GCE modified electrode;

s3, continuously dripping 5-8 mu L of 10 mu g/mL collagen antibody solution on the surface of the electrode prepared in the step S2, airing at room temperature, washing away unfixed antibody by using PBS (phosphate buffered saline) buffer solution with the pH of 7.4 to obtain anti-III-Col/ReS₂a/GCE modified electrode;

s4, mixing anti-III-Col/ReS₂Cross-linking the/GCE modified electrode in glutaraldehyde steam for 5min, washing with PBS buffer solution with pH of 7.4, and air drying at room temperature;

s5, soaking the modified electrode prepared in the step S4 in 0.25 wt% bovine serum albumin solution for 0.5h to block non-specific binding sites possibly existing on the surface of the electrode, taking out the electrode, washing the electrode with PBS (phosphate buffered saline) with the pH of 7.4, and airing the electrode at room temperature to obtain anti-III-Col (BSA)/ReS₂the/GCE modified electrode, namely the collagen type III photoelectrochemical sensor, is placed in a refrigerator at 4 ℃ for storage for later use.

The invention also provides application of the triple-collagen photoelectrochemical sensor prepared by the method and constructed based on the rhenium disulfide nanosheet in qualitative and/or quantitative detection of triple-collagen antigens.

The method for detecting the collagen type III antigen by the collagen type III photoelectrochemical sensor constructed based on the rhenium disulfide nanosheet comprises the following steps:

a. preparing a standard solution: preparing a group of collagen antigen standard solutions with different concentrations including blank standard samples;

b. modification of a working electrode: the photoelectrochemical sensor prepared by the preparation method is used as a working electrode, and the working electrode is placed in the concentration prepared in the step a, wherein the concentration is respectively 0.0005, 0.001, 0.01, 0.1, 1, 10, 50, 100, 1000 ng.mL^-1The collagen III antigen standard solution is incubated to prepare III-Col-anti-III-Col (BSA)/ReS₂a/GCE modified electrode;

c. drawing a working curve: testing with three-electrode system of electrochemical workstation, and mixing III-Col-anti-III-Col (BSA)/ReS prepared in step b₂The method comprises the following steps of taking a/GCE modified electrode as a working electrode, a platinum sheet electrode as a counter electrode and a saturated calomel electrode as a reference electrode, and testing in a solution taking a phosphoric acid buffer solution of ascorbic acid as a supporting electrolyte; an i-t test means is adopted to draw a working curve according to the relation between the obtained photocurrent value and the concentration of the triple collagen antigen standard solution;

d. and testing the photocurrent value of the sample to be tested, and calculating the concentration of the triple collagen antigen in the sample to be tested by combining the working curve.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention successfully prepares 1-butyl-3-dodecyl imidazole bromide ionic liquid, then uses the ionic liquid as an auxiliary reagent, and uses a probe to strip rhenium disulfide powder by ultrasonic to successfully prepare a two-dimensional lamellar nano material with good biocompatibility and large specific surface area, and loads a collagen antibody based on a rhenium disulfide nanosheet modified electrode to successfully construct a collagen type photoelectrochemical sensor. The preparation method of the collagen type III photoelectrochemical sensor is simple, realizes the rapid, sensitive and high-selectivity detection of the collagen type III antigen in the sample, has low manufacturing cost, can be applied to portable detection, and has wide application prospect in the medical field.

(2) The invention firstly proposes that the 1-butyl-3-dodecyl imidazole bromide ionic liquid is used for stripping rhenium disulfide, and the long chain of the ionic liquid can extend into the ReS in the stripping process₂In the laminated structure to assist the electrostatic force between the laminated layers, thereby improving the ReS₂The peeling efficiency of (3); on the other hand, the ionic liquid can be attached to the ReS₂Surface of the sheet, thereby preventing the sheet ReS from being peeled off₂Re-polymerizing the agglomerates to increase the ReS₂Dispersibility and stability of the compound.

(3) The invention adopts a probe ultrasonic machine pair ReS₂Powder preparationStripping, the seismic source can be directly contacted with the rhenium disulfide solution, and the ultrasonic energy is more concentrated, thereby greatly shortening the ReS₂To increase the ReS₂Stripping efficiency and its photoelectrochemical activity.

(4) The invention applies the rhenium disulfide two-dimensional lamellar structure nano material to the preparation of the photoelectrochemical biosensor for the first time, because of the ReS₂The two-dimensional lamellar structure nano material is a transition metal disulfide with direct band gap property, thereby being beneficial to the separation of photogenerated electrons and holes, having good biocompatibility and large specific surface area, being beneficial to keeping the bioactivity of an antibody and improving the detection sensitivity.

(5) The ionic liquid has good conductivity, good biocompatibility and wide potential window, and the invention utilizes the ionic liquid as a stripping reagent to assist in stripping the transition metal disulfide, thereby being beneficial to keeping the biological activity of the antibody and simultaneously utilizing the ionic liquid as an electronic conductor to cooperatively improve the detection sensitivity of the sensor.

(6) The method adopts the ionic liquid as the stripping reagent, is green and environment-friendly, provides a new and wider development prospect for the application of the photoelectric material in the aspect of the electrochemical sensor, and has wider potential use value.

Drawings

FIG. 1 shows the NMR spectrum of the ionic liquid of 1-butyl-3-dodecyl imidazole bromide salt prepared in example 1.

FIG. 2 shows the ReS obtained in example 2₂Transmission electron microscopy characterization of the nanoplatelets.

FIG. 3 shows the ReS obtained in example 2₂Atomic force microscopy characterization of the nanoplatelets.

FIG. 4 shows the ReS obtained in example 2₂Ultraviolet-visible absorption spectrogram of the nanosheet.

In FIG. 5, (a) and (b) are ReS obtained in example 2₂And (3) analyzing the results of X-ray photoelectron spectroscopy analysis of rhenium and sulfur elements in the nanosheets.

FIG. 6 shows the ReS obtained in example 2₂And (5) performing X-ray powder diffraction characterization on the nanosheets.

FIG. 7 is a schematic diagram of the process for preparing the III-Col photoelectrochemical sensor of the present invention.

FIG. 8 is a graph of photocurrent response versus incubation time for a photoelectrochemical sensor of the present invention.

FIG. 9 is a graph of photocurrent response versus incubation temperature for a photoelectrochemical sensor of the present invention.

FIG. 10 is a graph of photocurrent response versus ascorbic acid concentration for a photoelectrochemical sensor of the present invention.

FIG. 11 shows naked GCE (curve a), ReS prepared according to the present invention₂(ii)/GCE (curve b), anti-III-Col/ReS₂(ii)/GCE (curve c), anti-III-Col (BSA)/ReS₂(ii)/GCE (Curve d) and III-Col-anti-III-Col (BSA)/ReS₂AC impedance plot of/GCE (curve e).

FIG. 12 shows naked GCE (curve a), ReS prepared according to the present invention₂(ii)/GCE (curve b), anti-III-Col/ReS₂(ii)/GCE (curve c), anti-III-Col (BSA)/ReS₂(ii)/GCE (Curve d) and III-Col-anti-III-Col (BSA)/ReS₂Photocurrent-time response curve of/GCE (curve e).

In FIG. 13, (a) and (b) are ReS, respectively₂Modified electrode, anti-III-Col/ReS₂Characterization of the interface scanning electron microscope of the modified electrode.

FIG. 14 (A) and (B) are a graph showing the photocurrent response of the photoelectrochemical sensor prepared in example 3 to different concentrations of III-Col antigen, and a linear relationship curve of the sensor to III-Col response, respectively.

Fig. 15 shows the experimental results of the anti-interference performance of the photoelectrochemical sensor manufactured by the present invention.

Fig. 16 is a result of experiment of photocurrent-time stability of the photoelectrochemical sensor manufactured according to the present invention.

In FIG. 17, (A) and (B) show ReS obtained in comparative example 1 and ReS obtained in example 2, respectively₂The concentration of the/GCE modified electrode is 0.1 mol.L^-10.1 mol. L of ascorbic acid^-1Graph of the results of the photoelectrochemical property test performed in PBS buffer (pH 7.0).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly apparent, the present invention is further described in detail with reference to the following embodiments; it should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention; reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

The white light lamp used in the following examples is a 5W high-brightness external white light source purchased from Taobao Shibao in light concept car light ornaments shop, and the website is as follows: http:// item. spm is a1z09.2.9.101.lFDGY0& id 39175152864& _ u fp3euhgb0aa & qq-pf-to pcqq.c2c, and the manufacturer is Huizhou Yunfeng Lighting science and technology Limited.

The light source emitted by the white light LED lamp is guided to the surface of the electrode through an optical fiber with the diameter of 8mm, and the light intensity of the light source is measured by a radiometer to be 180mW/cm²。

The present invention will be described in further detail below with reference to specific embodiments and with reference to the attached drawings.

Example 1

A1-butyl-3-dodecyl imidazole bromide ionic liquid is prepared by the following steps:

(1) weighing sodium hydride (0.04mol, 0.96g) and adding a small amount of sodium hydride into 120mL acetonitrile solution containing imidazole (0.02mol, 1.36g) for multiple times, carrying out ice-bath reaction for 3h to form white acetonitrile suspension of imidazole sodium, and cooling to room temperature for later use;

(2) adding bromododecane (0.01mol, 2.42g) into the acetonitrile suspension of the imidazole sodium obtained in the step (1), heating to 65 ℃, stirring overnight, monitoring the reaction process by a dot-matrix plate, performing suction filtration after the reaction is finished, removing insoluble impurities, performing rotary evaporation to remove acetonitrile to obtain a crude product, performing gradient elution by a silica gel column, wherein an eluent is (CH)₂Cl₂MeOH 10:1) to give dodecyl imidazole as a pale yellow oily liquid;

(3) dissolving the dodecyl imidazole (0.01mol, 2.36g) and n-butyl bromide (0.02mol, 2.74g) obtained in the step (2) in 40mL of acetonitrile, heating to 65 ℃, stirring overnight, monitoring the reaction process by a point plate, removing the acetonitrile by rotary evaporation after the reaction is completed to obtain a crude product, and performing gradient elution through a silica gel columnEluent is (CH)₂Cl₂MeOH 10:1) gave 1-butyl-3-dodecylimidazole bromide as a yellow viscous oil.

¹HNMR(400MHz，D₂O) δ: 9.55(s,1H),7.92(s,2H),4.20(dd, J ═ 12.7,6.9Hz,4H), 1.87-1.67 (m,4H), 1.43-1.10 (m,20H),0.85(t, J ═ 7.4Hz,3H),0.80(t, J ═ 6.7Hz, 3H). As shown in fig. 1, the kind and content of hydrogen atoms in the molecule can be determined by mapping the chemical shift value and integrating the peak area, thereby confirming that the structure of the product is correct.

Example 2

A preparation method of rhenium disulfide nanosheets based on ionic liquid assisted stripping comprises the following steps:

(1) taking 0.1g of 1-butyl-3-dodecyl imidazole bromide salt to a centrifuge tube, adding 15mL of N-methyl pyrrolidone to completely dissolve the 1-butyl-3-dodecyl imidazole bromide salt, and adding 20mg of ReS to the centrifuge tube₂Performing ultrasonic treatment on the powder by using a probe ultrasonic machine, wherein the ultrasonic treatment time of the probe is 3h, the power is 300W, the ultrasonic machine is set to work for 5s and pause for 2s, and the dispersed ReS is obtained₂A solution of nanoplatelets;

(2) the ReS obtained in the step (1)₂Subpackaging the nanosheets into centrifuge tubes, and centrifuging at 500rpm for 5min to remove blocky ReS₂Collecting the extract containing ReS₂Centrifuging the supernate of the nanosheets at 12000rpm for 15min to remove the solvent, alternately centrifuging and washing with deionized water and absolute ethyl alcohol for three times, and vacuum drying to obtain ReS₂Nanosheets.

FIG. 2 shows the ReS obtained in this example₂The transmission electron microscope characterization picture of the nano-sheet can be seen from the result in the picture, the ReS is obtained after ultrasonic stripping₂Is of a nano-platelet structure, and ReS₂The nano-sheets are uniformly dispersed and stacked to form a ReS with the size of about 100-300 nm₂And (4) nano flakes.

FIG. 3 shows the ReS obtained in this example₂Atomic force microscope characterization diagram of nanosheet, and from the diagram, it can be seen that the ReS prepared by the invention₂The thickness of the nano-sheet is 5-6 layers, and the lamellar structure is obvious.

FIG. 4 shows the ReS obtained in this example₂UV-visible of the nanoplatesAbsorption spectrum, from which it can be seen that ReS₂The nanoplatelets show strong absorption in the NIR region and a characteristic absorption peak at 812.5 nm. Thus illustrating the ReS produced by the present invention₂The nano-sheet has strong light absorption capacity, and is beneficial to improving the photoelectric conversion efficiency and the detection sensitivity of the sensor.

In FIG. 5, (a) and (b) are respectively ReS obtained in this example₂And (3) analyzing the results of X-ray photoelectron spectroscopy analysis of rhenium and sulfur elements in the nanosheets. 41.95eV and 44.35eV in the graph are ascribed to Re⁴⁺4f_7/2、Re⁴⁺4f_5/2Electron binding energy of (d); 162.4eV and 163.6eV are ascribed to Sulfur (S)^2-2p_3/2And S^2-2p_1/2) Two characteristic peaks of (a). Thus indicating that the invention successfully prepares the ReS₂Nanosheets.

FIG. 6 shows the ReS obtained in this example₂Characterization result of X-ray powder diffraction of nanosheet, ReS₂The nanosheet has four strong diffraction peaks at 2 theta values of about 14.6 degrees, 29.6 degrees, 44.9 degrees and 61.2 degrees, which correspond to ReS₂The (100), (002), (3-21) and (203) four crystal planes of the triclinic phase. The test results and pure triclinic ReS₂In accordance with JCPDS card No. 24-0922. Thus indicating that the invention successfully prepares the ReS₂Nanosheets.

Example 3

As shown in fig. 7, a method for constructing a collagen type III photoelectrochemical sensor based on a rhenium disulfide nanosheet includes the following steps:

s1, pretreating a glassy carbon electrode: straight bare glassy carbon electrode (diameter 3mm) using 0.3 μm Al₂O₃Polishing the suspension, washing with distilled water, and sequentially adding HNO₃Ultrasonically cleaning the mixture by using ethanol and secondary distilled water, and airing the mixture for later use;

s2 ReS obtained in example 2₂The nano-sheets are dispersed in water to form 2mg/mL^-1Taking 8. mu.L of ReS₂The dispersed liquid is dripped on the surface of the clean glassy carbon electrode obtained in the step S1 and dried at room temperature to prepare ReS₂a/GCE modified electrode;

S3、continuously dripping 6 μ L of 10 μ g/mL collagen type III antibody solution on the surface of the electrode prepared in step S2, air drying at room temperature, and adding 0.01mol/L^-1Washing unfixed antibody by PBS buffer solution with pH of 7.4 to obtain anti-III-Col/ReS₂a/GCE modified electrode;

s4, mixing anti-III-Col/ReS₂the/GCE modified electrode is crosslinked for 5min in glutaraldehyde steam by 0.01 mol.L^-1Washing with PBS buffer solution with pH 7.4, and air drying at room temperature;

s5, soaking the modified electrode prepared in the step S4 in 0.25 wt% bovine serum albumin solution for 0.5h to block non-specific binding sites possibly existing on the surface of the electrode, taking out the electrode, washing the electrode with PBS (phosphate buffered saline) with the pH of 7.4, and airing the electrode at room temperature to obtain anti-III-Col (BSA)/ReS₂A GCE modified electrode, namely a collagen type III photoelectrochemical sensor, is placed in a refrigerator at 4 ℃ for storage for later use;

s6, anti-III-Col (BSA)/ReS₂the/GCE modified electrodes are respectively placed in III-Col antigen solutions with different concentrations, and the concentrations are respectively as follows: 0.0005, 0.001, 0.01, 0.1, 1, 10, 50, 100, 1000 ng/mL^-1Incubating at 35 ℃ for 25min, then incubating with 0.01 mol. L^-1Washing with PBS buffer solution with pH of 7.4 to obtain III-Col-anti-III-Col (BSA)/ReS₂the/GCE modified electrode is a photoelectrochemical sensor of III-Col combined collagen type III, and is stored at 4 ℃ for later use.

S7, self-assembly photoelectrochemistry test system: A5W white light lamp is used as an excitation light source and is guided to the surface of an electrode through an optical fiber, and a three-electrode system is adopted to test photocurrent: Hg/Hg with a 3mm straight glassy carbon electrode as the working electrode₂Cl₂The electrode (saturated KCl) was used as a reference electrode and a platinum wire electrode as a counter electrode, and the photocurrent was measured by CHI660E electrochemical workstation (shanghai chenhua instruments). The photocurrent was at a constant potential (0.05V vs Hg/Hg)₂Cl₂)、0.1mol·L^-10.1 mol. L of ascorbic acid^{- 1}In PBS buffer (pH 7.0).

Regarding step S6, the optimal incubation time for iii-Col antigen can be determined by examining the photocurrent response of the sensor versus the incubation time, and the results are shown in fig. 8. The results in the figure show that: within the range of 10-35 min, along with the extension of the incubation time, the current response difference value before and after the immunosensor recognizes III-Col is rapidly increased and tends to be stable after 25min, which indicates that the antigen-antibody immunological binding tends to be saturated, and therefore, 25min is selected as the optimal incubation time.

Regarding step S6, the optimal incubation temperature for iii-Col antigen can be determined by examining the photocurrent response of the sensor versus incubation temperature, and the results are shown in fig. 9. The results in the figure show that: within the range of 25-45 ℃, as the incubation temperature rises, the current response difference value before and after the immunosensor identifies III-Col is rapidly increased, and reaches the maximum value at 35 ℃; when the temperature is higher than 35 ℃, the activity of the protein is influenced, the recognition capability of the protein is reduced, and the current response difference is reduced. Therefore, the present invention selects 35 ℃ as the optimal incubation temperature.

In step S7, the optimum concentration of ascorbic acid is 0.1 mol.L of ascorbic acid at different concentrations by the same sensor^-1The photocurrent-time response curve in PBS buffer (pH 7.0) was determined and the results are shown in fig. 10. The results in the figure show that: from 0 to 0.10 mol.L^-1As the ascorbic acid concentration gradually increased, the photocurrent also gradually increased. As the ascorbic acid concentration further increased, the photocurrent decreased. This is because if the concentration is too large, the absorbance of ascorbic acid in the solution increases, which leads to a decrease in the light intensity irradiated to the electrode surface and a decrease in the excitation efficiency of the quantum dot. Therefore, in view of the sensitivity of the electrode response to ascorbic acid concentration, the present invention selects 0.10 mol.L^-1As the optimum concentration of ascorbic acid.

The following photoelectrochemical property tests also all adopt the optimum parameter conditions determined above.

Example 4

As shown in fig. 11, the modified electrode prepared in each step of example 3 was characterized by electrochemical alternating current impedance spectroscopy (EIS), which is one of the effective tools for exploring the properties of chemically modified electrode interface. The spectrogram is generally divided into a high-frequency part and a low-frequency part, wherein the high-frequency part is a dynamic control area, and the low-frequency part is an expansion partA distributed control area. At 5.0 mmol. multidot.L^-1K₃[Fe(CN)₆]/K₄[Fe(CN)₆](1:1)+0.1mol·L^-1PBS(pH＝7.0)+0.1mol·L^-1Alternating current impedance characterization is carried out in a KCl solution, and charge transfer resistance of an electrode interface is calculated by using a Randlescirt fitting circuit, wherein the interface charge transfer resistance (Rct) and a diffusion resistance (ZW) are connected with an interface capacitance (Cdl) in parallel, and the diameter of a semicircle corresponds to the interface charge transfer resistance (Rct). The Nyquist curve is shown in fig. 11, and it can be seen that the half circle of the alternating-current impedance spectrum of the bare electrode (curve a) at the high-frequency part is very small, and the Rct of the alternating-current impedance spectrum is 68.0 Ω through simulation calculation; when ReS₂After the nano sheet is modified to the surface of the electrode, the semi-circle diameter of the high-frequency part of the nano sheet is increased (curve b), the impedance is larger, and the interface charge transfer resistance is increased to 1305.6 omega; curve c is anti-III-Col/ReS₂An alternating current impedance spectrogram of an interface of the modified electrode can block the electron transfer of the interface because the antibody is a biological macromolecule which is not beneficial to the electron transfer, and the charge transfer resistance of the interface is increased to 1337.6 omega; after blocking the non-specific active sites possibly existing on the surface of the electrode by BSA, the semi-circle diameter of an alternating current impedance spectrum is greatly increased (curve d), and the charge transfer resistance of the electrode is 4391.7 omega through simulation calculation; when the immunosensor is matched with 0.1 ng/mL^-1After the specific binding of the III-Col antigen, the antigen-antibody complex covers the electrode surface, and the interface charge transfer resistance of the antigen-antibody complex is further increased to 8178.8 omega (curve e). The results show that the III-Col photoelectrochemical sensor is successfully prepared after gradual modification.

As shown in FIG. 12, after the modified electrode surface is combined with anti-III-Col antibody (c) and BSA (d) to block inactive sites, ReS₂The photocurrent of the modified electrode was reduced from 14 μ A to 10.25 μ A and 7.23 μ A, respectively. This is probably due to the inhibition of electron transport by the protein, which makes it difficult for the electron donor ascorbic acid in solution to diffuse to the electrode surface, resulting in a decrease in photocurrent intensity. When the immunosensor is further mixed with 0.1 ng/mL^-1After specific binding of the III-Col antigen (e), a further reduction in photocurrent intensity to 5.86. mu.A was observed. Therefore, the III-Col photoelectrochemical sensor successfully prepared by the method is shown.

The interface during electrode modification process was characterized by scanning electron microscope, and in FIG. 13, (a) and (b) are respectively ReS₂Modified electrode, anti-III-Col (BSA)/ReS₂Modifying the interface of the electrode. As can be seen from the figure, when ReS₂After the modification is carried out on the surface of the electrode, the surface of the electrode is covered with a large number of ReS with a lamellar structure₂The nano material has the characteristic of large specific surface area, is favorable for improving the adsorption capacity and action sites of the antibody on the surface of the electrode, and is more favorable for electron conduction so as to improve the photoelectric conversion efficiency of the sensor; upon further modification of the III-Col antibodies, we found ReS₂The lamellar structure is covered by the III-Col antibody protein and the interface is blurred compared to that before the antibody is modified, probably due to the cross-linking between glutaraldehyde and the antibody. The results show that the immunosensor is successfully prepared by layer-by-layer modification.

The photocurrent response conditions of the photoelectrochemical sensor in example 3 to different concentrations of III-Col antigens are examined by adopting a photocurrent-time method, and in fig. 14, (A) and (B) are respectively a photocurrent response condition graph of the photoelectrochemical sensor prepared in example 3 to different concentrations of III-Col antigens and a linear relation curve of the sensor to III-Col responses. From the results in the figure, it can be seen that: when the concentration of the III-Col is 0.0005-1000 ng.mL^-1In time (iii), anti-III-Col (BSA)/ReS₂Photocurrent difference (delta I ═ I) before and after bonding of modified electrode to III-Col antigen₀-I) is well linear with the logarithm of the III-Col antigen concentration, the linear equation being: Δ I ═ 0.362logC_Ⅲ-Col(ng·mL^-1) +1.778(R ═ 0.996), detection limit was 0.1pg · mL^-1(S/N-3). This shows that the 1-butyl-3-dodecyl imidazole bromide ionic liquid-assisted stripping based ReS₂The photoelectrochemical sensor constructed by the nanosheets can be used for high-sensitivity detection of III-Col antigens.

Example 5

The anti-interference performance is one of important indexes for measuring the practicability of the photoelectrochemical sensor. In order to examine the specific recognition performance of the III-Col photoelectrochemical sensor prepared in the embodiment 3 of the invention, carcinoembryonic antigen (CEA) is selected in the embodiment,five tumor markers of neuron-specific enolase (NSE), human serum albumin (HAS), Alpha Fetoprotein (AFP) and Squamous Cell Carcinoma Antigen (SCCA) were used as interferents for the selectivity experiments. Mixing 0.1 ng/mL^-1Respectively mixing with 10 ng/mL^-1The CEA, NSE, HAS, AFP and SCCA solutions are mixed and subjected to photoelectrochemical tests under the optimal conditions, and the results are shown in FIG. 15. The results in the figure show that: the photocurrent response difference of the immunosensor before and after the interferent is added has no obvious change, so that the photoelectrochemical sensor prepared by the method has good selectivity.

In order to further examine the repeatability of the III-Col photoelectrochemical sensor prepared by the invention, six anti-III-Col (BSA)/ReS are prepared at the same time₂(ii)/GCE, for a test concentration of 0.1 ng-mL^-1The photocurrent of the III-Col antigen solution of (1) was varied. The relative standard deviation of the measurement results of the 6 sensors is calculated to be 3.7%, which shows that the photoelectric sensor prepared by the invention has good repeatability.

To investigate the stability of the prepared immunosensor. The prepared sensor was placed in a phosphate buffer solution with pH 7.4, stored in a refrigerator at 4 ℃ for further use, and used to measure a concentration of 0.1 ng/mL after 2 weeks^-1III-Col antigen solution photocurrent change. Experiments show that the photocurrent response difference value is 91.6% of the initial value, which indicates that the immunosensor has good stability and can better maintain the biological activity of the antibody.

In addition, to further examine the anti-III-Col (BSA)/ReS prepared₂The stability of the electrode is modified by the/GCE. The time of each illumination is about 10s by controlling the on/off of the LED light source, and the electrodes are continuously excited more than 16 times in 10 minutes, and the result is shown in fig. 16. From the results in the figure, it can be seen that: the photocurrent signal is stable, and almost no obvious photocurrent attenuation phenomenon exists. The modified electrode is shown to be 0.10 mol.L^-1The ascorbic acid solution has very good stability, and can be used as an immunosensor.

Example 6

In order to examine the practical application value of the photoelectrochemical sensor prepared by the invention, the photoelectrochemical sensor is usedThe content of III-Col in clinical serum samples is detected. In the experiment, 2 clinical patients with abdominal aortic aneurysm are selected, serum samples of the patients before and after operation are collected, and the serum samples are diluted by PBS (phosphate buffer solution) with the pH value of 0.01mol/L being 7.4 according to the proportion of 1:50 and then used as samples to be tested to test anti-III-Col (BSA)/ReS prepared by the invention₂Practicality of the/GCE sensor, 3 measurements per blood sample are given in table 1 below.

TABLE 1 results of III-Col assay in clinical serum samples

From the above table results, it can be seen that: after patients A and B receive surgery, the level of III-Col in serum is increased, which shows that the invention can play a positive role in patients with abdominal aortic aneurysm through surgical treatment, and the determination result is accurate and reliable, so that the invention is expected to be used in clinical screening and diagnosis of abdominal aortic aneurysm, and has wide potential application value.

Comparative example 1

The comparative example provides a preparation method of rhenium disulfide nanosheet based on ionic liquid assisted stripping, and compared with example 2, the differences are that: directly mix 20mg of ReS₂Uniformly dispersing the powder in 15mL of N-methylpyrrolidone, performing ultrasonic treatment by using a probe ultrasonic machine, wherein the ultrasonic treatment time of the probe is 3h, the power is 300W, and the ultrasonic machine is set to work for 5s and pause for 2s to obtain the dispersed ReS₂A solution of nanoplatelets. The rest is the same as the embodiment 2, and the description is omitted.

The ReS prepared in the comparative example was added₂Dispersing the nano-sheet in water to form 2mg/mL dispersion, and collecting 8 μ L of ReS₂The dispersed liquid is dripped on the surface of the clean glassy carbon electrode obtained in the step S1 and dried at room temperature to prepare ReS₂the/GCE modified electrode.

The ReS prepared in the comparative example and example 2₂The concentration of the/GCE modified electrode is 0.1 mol.L^-10.1 mol. L of ascorbic acid^-1The photoelectrochemical properties were measured in a PBS buffer (pH 7.0) and the results are shown in fig. 17 (a) and (B), respectively. The results can be seen from the figureIn the invention, the ReS prepared by taking 1-butyl-3-dodecyl imidazole bromide ionic liquid as stripping reagent for auxiliary stripping is adopted₂The photoelectric current value of the nano-sheet is 14 muA, which is obviously higher than that of ReS prepared by stripping with the traditional method₂The photocurrent value of the nanosheets was 9 μ a. Therefore, the invention shows that the ReS is stripped under the assistance of the 1-butyl-3-dodecyl imidazole bromide ionic liquid₂Nano material capable of raising ReS obviously₂The stripping efficiency and the photoelectrochemical properties of the film.

While the invention has been described with respect to specific embodiments thereof, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention; those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and alterations of the above embodiments according to the spirit and techniques of the present invention are also within the scope of the present invention.

Claims (10)

1. A preparation method of rhenium disulfide nanosheets based on ionic liquid assisted stripping is characterized by comprising the following steps: dissolving 1-butyl-3-dodecyl imidazole bromide salt in N-methyl pyrrolidone, and adding ReS₂Subjecting the powder to ultrasonic treatment with a probe ultrasonic machine to obtain a dispersion of the ReS₂Centrifuging, washing and drying the solution of the nanosheets to obtain the ReS₂Nanosheets.

2. The preparation method of rhenium disulfide nanosheet based on ionic liquid assisted stripping as claimed in claim 1, wherein the preparation method of 1-butyl-3-dodecyl imidazole bromide salt comprises the following steps:

(1) adding a small amount of sodium hydride into an acetonitrile solution containing imidazole for multiple times, reacting in ice bath to obtain an acetonitrile suspension of white imidazole sodium, and cooling to room temperature for later use;

(2) adding bromododecane into the suspension, stirring at reflux temperature overnight, performing suction filtration after the reaction is finished, taking the filtrate, and performing spin-drying on the solvent to obtain a crude product, and purifying to obtain a light yellow oily liquid dodecyl imidazole bromide salt;

(3) and (3) dissolving the dodecyl imidazole bromide salt and n-butyl bromide obtained in the step (2) in acetonitrile, stirring at a reflux temperature overnight, drying the solvent in a spinning mode after the reaction is finished to obtain a crude product, and purifying to obtain the yellow viscous oily liquid 1-butyl-3-dodecyl imidazole bromide salt.

3. The preparation method of rhenium disulfide nanosheet based on ionic liquid assisted stripping as claimed in claim 2, wherein the molar ratio of sodium hydride, imidazole, and bromododecane is 4:2: 1.

4. The preparation method of rhenium disulfide nanosheet based on ionic liquid assisted stripping as claimed in claim 2, wherein in step (3), the molar ratio of the dodecyl imidazole bromide salt to n-butyl bromide is 1: 1-3.

5. The preparation method of rhenium disulfide nanosheet based on ionic liquid assisted stripping as claimed in claim 1, wherein the 1-butyl-3-dodecyl imidazole bromide salt and the ReS₂The dosage ratio of the powder is 2-8: 1.

6. The preparation method of the rhenium disulfide nanosheet based on ionic liquid assisted stripping as claimed in claim 1, wherein the probe ultrasonic treatment time is 2-4 h, the power is 300W, and the ultrasonic machine is set to work for 5s and pause for 2 s.

7. The preparation method of the rhenium disulfide nanosheet based on ionic liquid assisted stripping as claimed in claim 1, wherein the centrifugation is performed for 5min at 500rpm and then for 15min at 12000 rpm.

8. A method for constructing a collagen type III photoelectrochemical sensor based on a rhenium disulfide nanosheet is characterized by comprising the following steps:

s1, pretreating a glassy carbon electrode;

s2, taking 6-12 mu L of ReS prepared by any one of claims 1-7₂The nano sheet dispersion liquid is dripped on the surface of the clean glassy carbon electrode obtained in the step S1 and dried at room temperature to prepare ReS₂a/GCE modified electrode;

s3, continuously dripping 5-8 mu L of 10 mu g/mL collagen antibody solution on the surface of the electrode prepared in the step S2, airing at room temperature, washing away unfixed antibody by using PBS (phosphate buffered saline) buffer solution with the pH of 7.4 to obtain anti-III-Col/ReS₂a/GCE modified electrode;

s4, mixing anti-III-Col/ReS₂Cross-linking the/GCE modified electrode in glutaraldehyde steam for 5min, washing with PBS buffer solution with pH of 7.4, and air drying at room temperature;

s5, soaking the modified electrode prepared in the step S4 in 0.25 wt% bovine serum albumin solution for 0.5h to block non-specific binding sites possibly existing on the surface of the electrode, taking out the electrode, washing the electrode with PBS (phosphate buffered saline) with the pH of 7.4, and airing the electrode at room temperature to obtain anti-III-Col (BSA)/ReS₂the/GCE modified electrode, namely the collagen type III photoelectrochemical sensor, is placed in a refrigerator at 4 ℃ for storage for later use.

9. Use of the triple collagen photoelectrochemical sensor constructed based on the rhenium disulfide nanosheets prepared by the method according to claim 8 in qualitative and/or quantitative detection of triple collagen antigens.

10. The application of the triple-type collagen photoelectrochemical sensor constructed based on the rhenium disulfide nanosheets according to claim 9, wherein the method for detecting the triple-type collagen antigens comprises the following steps:

a. preparing a standard solution: preparing a group of collagen antigen standard solutions with different concentrations including blank standard samples;

b. modification of a working electrode: taking the photoelectrochemical sensor prepared by the preparation method of claim 7 as a working electrode, and placing the working electrode in the collagen III antigen standard solutions with different concentrations prepared in the step a for incubation to prepare III-Col-anti-III-Col (BSA)/ReS₂a/GCE modified electrode;

c. drawing a working curve: testing with three-electrode system of electrochemical workstation, and mixing III-Col-anti-III-Col (BSA)/ReS prepared in step b₂The method comprises the following steps of taking a/GCE modified electrode as a working electrode, a platinum sheet electrode as a counter electrode and a saturated calomel electrode as a reference electrode, and testing in a solution taking a phosphoric acid buffer solution of ascorbic acid as a supporting electrolyte; an i-t test means is adopted to draw a working curve according to the relation between the obtained photocurrent value and the concentration of the triple collagen antigen standard solution;

d. and testing the photocurrent value of the sample to be tested, and calculating the concentration of the triple collagen antigen in the sample to be tested by combining the working curve.

Images