CN109283235B

CN109283235B - Based on NSCQDs/Bi2S3Photoelectrochemical sensor and preparation method thereof

Info

Publication number: CN109283235B
Application number: CN201811347164.6A
Authority: CN
Inventors: 高文华; 成文杰; 陈耀文; 张晓珊; 林月娟; 徐严平; 陈图锋
Original assignee: Shantou University
Current assignee: Shantou University
Priority date: 2018-11-13
Filing date: 2018-11-13
Publication date: 2020-11-27
Anticipated expiration: 2038-11-13
Also published as: CN109283235A

Abstract

The invention relates to a method based on NSCQDs/Bi₂S₃The photoelectrochemical sensor and the preparation and the application thereof, and Bi₂S₃The nano-rod is an optical active material, and NSCQDs are synthesized by a PDDA electrostatic adsorption hydrothermal method; then theCarboxyl groups on the surfaces of NSCQDs are activated by EDC and NHS, and Ab is modified on the surfaces of the electrodes through amidation reaction. In the presence of AFP, an Ab-AFP immune complex is formed on the surface of an electrode through antigen-antibody recognition, so that the steric hindrance of the surface of the electrode is increased, the transmission of an electron sacrificial agent Ascorbic Acid (AA) to the surface of the electrode is hindered, and the change of a sensor photocurrent signal is caused. And detecting the AFP concentration according to the relation between the AFP concentration and the sensor photocurrent. The photoelectrochemical sensor provided by the invention has good selectivity, repeatability and stability. The invention has the advantages of simple ATP detection operation, low cost, high efficiency and high sensitivity, lower detection limit and wider detection range, and has important significance for clinical and biological analysis of protein.

Description

Based on NSCQDs/Bi2S3Photoelectrochemical sensor and preparation method thereof

Technical Field

The invention relates to the technical field of photoelectrochemical sensor detection, in particular to a method based on NSCQDs/Bi₂S₃The photoelectrochemical sensor and the preparation and the application thereof.

Background

With the progress and development of society, people are more concerned about environmental protection, health and safety and the like. Currently, cancer is one of the major threats to humans and their health. Therefore, detection of cancer markers plays an important role in early screening and disease diagnosis. Alpha-fetoprotein (AFP) is a human plasma protein encoded by the AFP gene and produced by the yolk sac and liver during the fetus. AFP is widely recognized as a cancer marker, and if AFP is present at high concentrations in the plasma of adults, it may be associated with the pathogenesis of certain cancers, such as hepatocellular carcinoma, endodermal cancer, testicular cancer, teratoma, yolk sac cancer, ovarian cancer, gastric cancer, and the like. Thus, the detection of AFP plays an important role in the early stage screening and prevention of cancer. The current detection methods for AFP include radioimmunoassay, enzyme-linked immunosorbent assay, electrochemiluminescence assay, fluorescence detection, etc. Although these methods can achieve sensitive detection of AFP, they require a lot of manpower and material resources, long detection time, and low efficiency. Therefore, there is a need to find other methods to achieve a fast, sensitive and efficient detection of AFP.

As a novel detection means, the photoelectrochemistry biosensor has attracted extensive attention due to the characteristics of a detection time period, high detection efficiency and the like. The photoelectrochemical biosensor involves a charge transfer process between the analyte, the photoactive material and the electrode under certain lighting conditions. Compared with optical and electrochemical luminescence detection methods, due to the separation of the excitation light source and the detection signal, the background noise is reduced to a great extent, and the detection sensitivity is improved. Therefore, the photoelectrochemical biosensor has been applied to the detection of various biological targets, such as DNA, protein small molecules, and the like. Active materials play a very important role in the construction of photoelectrochemical biosensors, and many photoactive materials have been used, for example, ZnO, TiO₂、SnO₂BiOI, and the like. Bismuth sulfide (Bi)₂S₃) As a chalcogenide metal oxide, which possesses a relatively narrow band gap, good absorption coefficient and photoelectric conversion efficiency, it has been applied to photodetectors, solar cells, photocatalysis, and the like. But Bi₂S₃The visible light absorption range of the light source is 300-510nm, and the light absorption of the light source is weak for 510-800 nm. Carbon Quantum Dots (CQDs) as a novel carbon-based material have characteristics of good light absorption capacity, high fluorescence efficiency, and the like, and also have nontoxicity, biocompatibility, high stability, and good water solubility, and have been applied in many fields.

Disclosure of Invention

The invention aims to provide a method based on NSCQDs/Bi₂S₃The photoelectrochemical sensor and the preparation and the application thereof solve the problems in the prior art.

In order to solve the problems, the invention discloses a method based on NSCQDs/Bi₂S₃The preparation of the photoelectrochemical sensor mainly comprises the following steps:

(1) preparation of Bi₂S₃A nanorod;

(2) synthesizing NSCQDs;

(3) adding Bi₂S₃Adding the nano-rod into deionized water, and obtaining Bi after ultrasonic dispersion₂S₃Dripping the suspension on an ITO conductive substrate, drying in an oven at 60 ℃ for 20-40min, taking out, and naturally cooling to room temperature to obtain Bi₂S₃An ITO modified electrode;

(4) bi obtained in step (3)₂S₃Dripping a PDDA (poly-diallyldimethylammonium chloride) solution containing NaCl on the surface of the ITO modified electrode, standing at room temperature for 10-20min, cleaning the electrode with deionized water, and then drying with nitrogen;

(5) measuring NSCQDs solution, dripping on the surface of the electrode, drying the electrode in a 60 ℃ oven for 30-50min, washing the electrode with deionized water, and drying at room temperature to obtain NSCQDs/Bi₂S₃An ITO modified electrode;

(6) the NSCQDs/Bi obtained in the step (5) are mixed₂S₃The ITO modified electrode is immersed in a solution containing EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), activated for 1h at room temperature, and then washed with PBS solution to wash away the excess EDC and NHS;

(7) ab (AFP antibody) solution is dripped in NSCQDs/Bi₂S₃ITO surface, and placing the ITO surface in a refrigerator at 4 ℃ for incubation for 6 h; after incubation was complete, the electrodes were washed with PBS solution to remove physisorbed abs; then, adding BSA solution dropwise to the surface of the electrode, placing the electrode in a refrigerator at 4 ℃, and placing for 2 hours to block off the active sites of non-specific binding; the electrodes were then washed with PBS solution.

Dropping PDDA to adsorb B by static electricity_i2S₃The ITO modified electrode surface can be used for layer-by-layer assembly to modify subsequent NSCQDs. The room temperature was allowed to stand for 15min in order to sufficiently adsorb PDDA by electrostatic interaction.

Carboxyl groups on NSCQDs react with carbodiimide to form an intermediate product of addition, and then react with amino groups on subsequent proteins to form amide bonds.

Further, Bi in the step (1)₂S₃The preparation of the nano-rod mainly comprises the following steps: weighing Bi (NO)₃)·5H₂Stirring in glycol to form transparent solution A; weighing Na₂S·9H₂Placing the O in ethylene glycol and deionized water, and stirring to form a transparent solution B; dropwise adding the solution B into the solution A under the condition of strong stirring; then adding urea and deionized water into the solution, stirring strongly, transferring the solution into a reaction kettle, and placing the reaction kettle at 180 ℃ for reaction for 24 hours; after the reaction is finished, naturally cooling to room temperature, washing the obtained black product with water and alcohol, and finally placing the black product in an oven at 60 ℃ for 8 hours.

Further, the synthesis of the NSCQDs in the step (2) mainly comprises the following steps: weighing ammonium citrate and thiourea, placing the ammonium citrate and the thiourea in deionized water, violently stirring to dissolve the ammonium citrate and the thiourea, transferring the ammonium citrate and the thiourea into a reaction kettle, placing the reaction kettle in an oven, and reacting for 4 hours at 160 ℃; after the reaction is finished, naturally cooling to room temperature, centrifuging the obtained solution to remove precipitates, and then transferring the solution to a dialysis bag for dialysis for 48 hours; followed by rotary evaporation and freeze drying.

Further, Bi is described in the step (3)₂S₃The concentration of the suspension was 3.0 mg/mL^-1。

Further, the concentration of the NSCQDs solution in the step (5) is 2.0 mg.mL^-1。

Further, the Ab solution in the step (7) has a concentration of 30. mu.g.mL^-1。

Further, the PBS concentration was 0.1M and pH 7.4.

NSCQDs/Bi-based prepared by the method₂S₃Is photoelectrochemizedA chemical sensor.

The above-mentioned NSCQDs/Bi-based₂S₃The use of a photoelectrochemical sensor of (1) for the quantitative detection of AFP.

The quantitative detection of AFP mainly comprises the following steps: dropping AFP solution with different concentration in BSA/Ab/NSCQDs/Bi₂S₃The ITO electrode surface is incubated for 1h at room temperature, and then the electrode is cleaned by PBS solution; the obtained immunosensing electrode is taken as a working electrode, an Ag/AgCl electrode is taken as a reference electrode, a platinum wire electrode is taken as a counter electrode, and a photocurrent signal is tested in a PBS solution containing 0.15M AA (ascorbic acid); under the condition of no bias voltage, an optical filter (lambda is more than or equal to 420nm) is adopted to filter exciting light, and the illumination intensity is 20mW cm^-2And light irradiation/light blocking is carried out at intervals of 10s, and a photocurrent test is carried out.

Bi of the present invention₂S₃Has good absorption for the light with the visible wavelength range of 300-510nm, and the absorption intensity for the light with the wavelength range of 510-800nm is continuously reduced. The NSCQDs have good up-conversion fluorescence capability, can convert light with the wavelength of 510-800nm into light with the wavelength range of 444.6-469nm, and the part of the up-conversion light can be Bi-converted₂S₃And (4) utilizing. Under the irradiation of visible light, Bi₂S₃And generating electron-hole pairs, wherein electrons can be directly transferred to the ITO electrode on one hand, and electrons can be firstly transferred to NSCQDs and then transferred to the ITO electrode on the other hand, and holes react with the electron sacrificial agent AA to inhibit the recombination of the electron-hole pairs. In addition, the upconversion fluorescence of NSCQDs can be converted by Bi₂S₃By using the method, the utilization rate of the visible light by the sensor is improved.

Meanwhile, consider NCQDs and Bi₂S₃After being compounded, the compound has the absorption capacity for visible light compared with pure Bi₂S₃Is improved. NSCQDs/Bi₂S₃Has better absorption for light with the wavelength range of 300-550nm, and continuously weakens the absorption for 550-800 nm. Under visible light conditions, NCQDs/Bi₂S₃Generating photo-generated electron-hole pairs, transferring electrons to ITO, reacting the holes with an electron sacrificial agent AA in the solution, and inhibiting the recombination of the electron-control pairs. Likewise, NSThe up-conversion fluorescence capability of CQDs can convert light with the wavelength range of 550-800nm into light with the wavelength range of 444.6-469.6nm, thereby improving the NSCQDs/Bi₂S₃Utilization ratio of visible light.

Since Bi₂S₃Matched with positions of CB and VB of NSCQDs, and Bi is used under the condition of illumination₂S₃And NSCQDs can generate electron-hole pairs, and electrons can be transferred from CB of NSCQDs to Bi₂S₃And then transferred to the ITO electrode; with holes from Bi₂S₃The VB of (a) is transferred to the VB of NSCQDs, which in turn reacts with the electron sacrificial agent AA to promote the separation of photogenerated electron-hole pairs.

In the presence of the detection target substance AFP, steric hindrance on the surface of the electrode is increased due to immunological recognition of Ab and AFP on the surface of the electrode, and transmission of AA to the surface of the electrode is blocked, so that the separation efficiency of photo-generated electron-hole pairs is reduced, and a photocurrent signal is reduced. Therefore, sensitive detection of AFP can be achieved based on the relationship between AFP concentration and sensor photocurrent.

Compared with the prior art, the invention adopts ammonium citrate and thiourea to prepare NSCQDs, and uses NSCQDs/Bi₂S₃The AFP antibody (Ab) is a biological recognition element, and the photoelectrochemistry biological immunosensor is constructed. Bi₂S₃The nano rod is used as a photoactive material, has good photoactivity and can provide stable optical signal output; the Eg of NSCQDs is 1.16eV, the utilization rate of visible light is high, the conductivity is good, and the transfer rate of photo-generated electrons is improved; in Bi₂S₃After being compounded with NSCQDs, on one hand, the modified electrode has improved conductivity and the transfer rate of photo-generated electrons is improved, and on the other hand, due to the unique up-conversion fluorescent property of the NSCQDs, the modified electrode can convert the light with the wavelength range of 600-900nm into the light with the wavelength range of 447-459 nm, thereby improving Bi₂S₃The utilization rate of visible light is improved, so that the output photocurrent signal is improved. Through the specific immune reaction between Ab and AFP, the immunosensor shows wide detection range and detection limit of the ground, and can realize the detection of cancerSensitive detection of the marker AFP. The invention also selects AA with a certain concentration as an electron sacrificial agent, thereby effectively promoting the separation of the photo-generated electron hole pairs. The photoelectrochemical sensor provided by the invention has good selectivity, repeatability and stability. The invention has the advantages of simple ATP detection operation, low cost, high efficiency and high sensitivity, lower detection limit and wider detection range, and has important significance for clinical and biological analysis of protein.

Drawings

FIG. 1 shows NSCQDs/Bi-based samples of the present invention₂S₃Schematic illustration of the preparation of a photoelectrochemical sensor of (a);

FIG. 2 shows Bi prepared according to the present invention₂S₃Scanning electron microscope image of the nano rod;

FIG. 3 is a transmission electron microscope image of NSCQDs prepared by the present invention, with the particle size distribution diagram shown in the inset;

FIG. 4 is a fluorescence spectrum and up-conversion fluorescence spectrum of NSCQDs of the present invention; (A) the fluorescence spectrum of NSCQDs, the excitation wavelength is 300nm to 440nm, and the increment is 10 nm; (B) the up-conversion fluorescence spectrum of the NSCQDs has the excitation wavelength of 500nm to 880nm and the increment of 20 nm;

FIG. 5 is an X-ray diffraction pattern of the present invention; wherein a is Bi₂S₃The nano-rods, b are NSCQDs; c is NSCQDs/Bi₂S₃；

FIG. 6 is a normalized UV-visible absorption spectrum of the present invention, wherein a is Bi₂S₃The nano-rod, b is NSCQDs/Bi₂S₃(ii) a The inset is the absorption spectrum of ultraviolet and visible light, a is Bi₂S₃The nano-rod, b is NSCQDs/Bi₂S₃；

FIG. 7 shows Bi of the present invention₂S₃(a) And NSCQDs/Bi₂S₃(b) Curve of (α h v) 1/2 versus photon energy (hv);

FIG. 8 is a Cyclic Voltammetry (CV) curve of NSCQDs of the present invention;

FIG. 9 shows (a) Bi in 0.1M PBS (pH 7.4) according to the present invention₂S₃ITO electrode and (b) NSCQD/Bi₂S₃Open circuit potential curve (OCP) of ITO electrode;

FIG. 10 shows the present invention in the presence of 5mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-Electrochemical Impedance Spectroscopy (EIS) of (a) and Cyclic Voltammetry (CV) curves of (B) different modified electrodes, and (C) photocurrent signals of different modified electrodes in PBS (0.1M, pH 7.4) solution containing 0.15M AA, under unbiased voltage under illumination (λ ≥ 420 nm): (a) ITO, (b) Bi₂S₃/ITO、(c)NSCQDs/Bi₂S₃/ITO、(d) Ab/NSCQDs/Bi₂S₃/ITO、(e)BSA/Ab/NSCQDs/Bi₂S₃ITO and (f) AFP/BSA/Ab/NSCQDs/Bi₂S₃/ITO；

FIG. 11(A) shows different AFP concentrations (a-h) of 0.001, 0.01, 0.1, 1.0, 10, 100, 300, 500 ng.mL^-1Current signals of the lower pair of photoelectrochemical biosensors; (B) a plot of AFP concentration versus corresponding photocurrent; B) the inset is a linear relationship between the logarithm of the AFP concentration and the sensor photocurrent signal;

FIG. 12 shows different concentrations of Bi (A) in PBS (0.1M, pH 7.4) containing 0.15M AA according to the present invention₂S₃For Bi₂S₃Influence of photocurrent of the ITO electrode; (B) different concentrations of AA to Bi in PBS (0.1M, pH 7.4)₂S₃Influence of photocurrent of the ITO electrode; (C) NSCQDs vs. NSCQDs/Bi at various concentrations in PBS containing 0.15M AA (0.1M, pH 7.4)₂S₃Influence of photocurrent of the ITO electrode; (D) PBS (0.1M) at various pH values containing 0.15M AA on NSCQDs/Bi₂S₃Influence of ITO electrode photocurrent;

FIG. 13 shows the incubation time of (A) different Ab concentrations and (B) different Ab/NSCQDs/Bi in PBS containing 0.15M AA (0.1M, pH 7.4) according to the invention₂S₃Influence of ITO electrode photocurrent (C) immunoreaction time vs. AFP/Ab/NSCQDs/Bi₂S₃Influence of ITO electrode photocurrent, AFP concentration is 10 ng-mL^-1；

FIG. 14 shows (b) Bi in the absence of light according to the present invention₂S₃ITO and (c) NSCQDs/Bi₂S₃Electrochemical resistance of/ITO, and (b') Bi under light irradiation₂S₃ITO and (c') NSCQDs/Bi₂S₃ITO electrochemical impedance; AFP concentration used in the test was 0.1 ng.mL^-1；

FIG. 15 shows (A)5 immunosensor electrode pairs 1 ng/mL in PBS containing 0.15M AA (0.1M, pH 7.4) under unbiased conditions with visible light irradiation (. lamda. gtoreq.420 nm)^-1The photocurrent signal of AFP detection of (A) and (B) the immunosensor pair after different storage times of 0.1 ng/mL^-1The photocurrent signal of AFP detection (C) the sensing electrode was coupled to a blank solution, 10 ng. mL ^-1 AFP solution, 100 ng.mL^-1CEA solution (100 ng. mL)^-1PSA solution (2), 100 ng. mL^-1BSA solution of (1), and 10 ng. mL^-1AFP, 100 ng.mL^-1CEA, 100 ng. mL^-1PSA of (2) and 100 ng.mL^-1The photocurrent signal of the BSA mixed solution.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Example 1

Based on NSCQDs/Bi₂S₃As shown in fig. 1, the preparation of the photoelectrochemical sensor mainly comprises the following steps:

1、Bi₂S₃preparation of nanorods

Preparation of Bi by hydrothermal method₂S₃And (4) nanorods. The method mainly comprises the following steps: 1.820g of Bi (NO) are weighed out₃)·5H₂Stirring O in 25mL of ethylene glycol for 10min to form a transparent solution A. 1.325g of Na was weighed₂S·9H₂And placing the solution O in 15mL of ethylene glycol and 15mL of deionized water, and stirring for 10min to form a transparent solution B. The solution B was added dropwise to the solution a with vigorous stirring, resulting in a large amount of black suspension. Then 1.922g of urea and 15mL of deionized water are added into the solution, the mixture is stirred vigorously for 20min and then transferred into a polytetrafluoroethylene reaction kettle, and the reaction kettle is placed at 180 ℃ for reaction for 24 h. After the reaction is finished, the reaction kettle is naturally cooled to room temperature, the obtained black product is washed by water and alcohol for 5 times respectively, and finally the black product is placed in a baking oven at 60 ℃ for 8 hours to obtain Bi₂S₃Nano-solidPowder, Scanning Electron Microscope (SEM) to observe the synthesized Bi₂S₃The size and morphology of (A) are shown in FIG. 2, Bi₂S₃Is a distinct rod-like structure with a diameter in the range of 20-60nm and a length between 50-140 nm.

2. Synthesis of NSCQDs

0.21g of ammonium citrate and 0.23g of thiourea are weighed and placed in 10mL of deionized water, stirred vigorously for 10min to dissolve the ammonium citrate and the thiourea, then the mixture is transferred into a polytetrafluoroethylene reaction kettle and placed in an oven to react for 4h at the temperature of 160 ℃. After the reaction was completed, the reaction vessel was allowed to cool naturally to room temperature, the resulting solution was centrifuged at 10000r/min for 10min, and then the precipitate was removed, and the resulting solution was transferred to a dialysis bag (MWCO ═ 1000) and dialyzed for 48 h. The resulting solution was subjected to rotary evaporation and freeze drying to obtain NSCQDs powder. Transmission Electron Microscopy (TEM) of NSCQDs, as shown in FIG. 3, the prepared NSCQDs have an approximately spherical structure, a particle size distribution in the range of 1.51-3.95nm, and an average size of 2.68 nm. The height of NCQDs was analyzed by an Atomic Force Microscope (AFM), and the height was 3nm or less. It is also found by energy dispersive X-ray spectroscopy and element area scan spectroscopy analysis of the NSCQDs that the prepared NSCQDs mainly contain C, N, O, S four elements, and the four elements are uniformly distributed. The structure and surface groups of the NSCQDs are characterized by Fourier infrared spectroscopy to obtain that the NSCQDs contain carboxyl functional groups.

The fluorescence spectrum and the up-conversion fluorescence spectrum of NSCQDs are shown in fig. 4 (a), (B), and show the property that the emission spectrum depends on the excitation wavelength, and when the excitation wavelength is increased from 300nm to 400nm, the emission peak is firstly shifted to the long wavelength, then shifted to the short wavelength, and finally shifted to the long wavelength; and the fluorescence emission intensity is reduced, increased and reduced at first. This phenomenon may be related to the particle size and surface defect state of NSCQDs. The optimal excitation wavelength is 400nm, the strongest emission peak position is 455nm, and the Stokes shift is 55 nm. And as can be seen from fig. 4, the prepared NSCQDs show obvious up-conversion fluorescence properties, the optimal up-conversion fluorescence excitation wavelength is 800nm, the maximum up-conversion fluorescence intensity peak position is 456nm, and the anti-Stokes shift is 344 nm.

3. ITO electrode cleaning and electrode modification preparation

And (3) cleaning an ITO electrode: ITO conductive substrate (3.0X 1.0 cm)²) The reaction mixture was washed with 1M NaOH (Vwater: Vacetone ═ 1: 1) the solution was sonicated for 30min, then washed three times with absolute ethanol and ultra pure water, respectively, and then dried in an oven at 105 ℃ for 45 min. And (4) placing the cleaned ITO conductive substrate in a shady and cool place for storage.

Weighing 15mg of Bi₂S₃Adding the solid into 3mL of deionized water, ultrasonically dispersing for 40min, dropwise adding 8 mu L of black suspension on an ITO conductive substrate, and fixing the area of the ITO conductive substrate to be 0.1256cm²Then placing the mixture in an oven at 60 ℃ for drying for 30min, taking out the dried mixture, and naturally cooling the dried mixture to room temperature to obtain Bi₂S₃the/ITO modifies the electrode.

In the obtained Bi₂S₃And dripping 8 mu L of 2M PDDA solution containing 0.5M NaCl on the surface of the ITO modified electrode, standing at room temperature for 15min, washing the electrode with deionized water, and drying with nitrogen. Then, 8. mu.L of 2 mg/mL was measured^-1Dropwise adding the NSCQDs solution on the surface of the electrode, drying the electrode in a 60 ℃ oven for 45min, carefully cleaning the electrode with deionized water, and drying at room temperature to obtain NSCQDs/Bi₂S₃the/ITO modifies the electrode.

4. Preparation of chemical biological immunosensor

The obtained NSCQDs/Bi₂S₃the/ITO-modified electrode was immersed in 1ml of a solution containing 20mg EDC and 10 mg NHS, activated for 1h at room temperature, and then carefully washed with a PBS (0.1M, pH 7.4) solution to wash off excess EDC and NHS. Then, 8. mu.L of 30. mu.g/ml was taken^-1The Ab solution is dripped into NSCQDs/Bi₂S₃ITO surface and incubated in a refrigerator at 4 ℃ for 6 h. After incubation was complete, the electrodes were washed with PBS (0.1M, pH 7.4) solution to remove physisorbed abs. Then, 8. mu.L of 0.1% BSA solution was added dropwise to the electrode surface, and the electrode was placed in a refrigerator at 4 ℃ and left for 2 hours to block off the non-specifically bound active sites. Then, the electrode was washed with a PBS (0.1M, pH 7.4) solution to obtainAn immunosensing electrode.

Performance testing

1. X-ray diffraction pattern

Prepared Bi₂S₃Nanorods (a), NSCQDs (b), and NSCQDs/Bi₂S₃(c) The X-ray diffraction pattern of (A) when NSCQDs are mixed with Bi as shown in FIG. 5₂S₃After compounding, the XRD spectrum and Bi thereof₂S₃The spectra are similar, which indicates that NSCQDs are similar to Bi₂S₃Bi is not changed after the composition₂S₃A crystalline form of (a). NSCQDs exhibit a broad diffraction peak at 2 θ ═ 24.56 °, and have a graphene-like structure.

2. Ultraviolet visible absorption spectrum

The light absorption property of the material is studied by using ultraviolet and visible light absorption spectrum, and as shown in curve a of FIG. 6, Bi₂S₃Shows good absorption for light with the wavelength range of 300-510nm, and the absorption intensity for light with the wavelength range of 510-800nm is continuously reduced; NSCQDs/Bi₂S₃The composite shows better absorption for light with the wavelength range of 300-550nm, and the absorption for 550-800nm is continuously reduced.

3. Optical bandgap calculation

Calculating the optical band gap according to Tauc curve, as shown in FIG. 7, and obtaining Bi by calculation₂S₃A band gap of 1.54eV, NSCQDs/Bi₂S₃The composite band gap is 1.37 eV. This indicates that NSCQDs are associated with Bi₂S₃After the composition, the band gap is reduced to some extent, and the utilization rate of visible light is improved.

According to the following formula, for Bi₂S₃The Valence Band (VB) and the Conduction Band (CB) of (C):

E_CB(eV)＝-χ+0.5Eg；

E_VB(eV)＝-χ-0.5Eg；

to obtain Bi₂S₃CB and VB of-5.18 eV and-6.72 eV, respectively.

The matching of the valence band conduction band of the optical active material plays an important role in the photoelectrochemical biosensing detection, and therefore, the band edge of NSCQDs is measured by adopting a cyclic voltammetry method. The test solution is acetonitrile solution containing 0.1M tetrabutylammonium hexafluorophosphate (TBAPF6) and saturated NSQCDs, a traditional three-electrode system is adopted, namely a glassy carbon electrode is taken as a working electrode, a platinum wire is taken as a counter electrode and an Ag/AgCl electrode is taken as a reference electrode, the solution is tested after being introduced with nitrogen for 15min, and the positions of a Conduction Band (CB) and a Valence Band (VB) of the NSQCDs are calculated according to the following formula:

E_CB/LUMO＝-(E_red+4.71)eV

E_VB/HOMO＝-(E_ox+4.71)eV；

wherein E is_redAnd E_oxRespectively, the initial reduction potential and the initial oxidation potential.

The results of the test are shown in FIG. 8, E for NSCQDs_redAnd E_oxCalculated as-0.38 eV and 0.36eV, and calculated as-4.33 eV and-5.07 eV for CB and VB to NSCQDs. Therefore, theoretically, the photogenerated electrons generated under the illumination condition of NSCQDs can be transferred to Bi₂S₃And thus, separation of the photo-generated electron-hole pairs can be promoted.

Thus, Bi can be obtained₂S₃The Eg of the nano rod is 1.54eV, so that the nano rod has good photoelectric activity; second Bi₂S₃The nanorods (semiconductor 1) can form type II heterojunctions with NSCQDs (semiconductor 2), with semiconductor 2 having CB and VB both located higher than semiconductor 1, with the steps in CB and VB oriented in the same direction. Due to the difference of chemical potentials between the

semiconductors

1 and 2, an energy band at the interface of the II-type heterojunction bends to generate a built-in field, so that a light-excited electron and a hole move in opposite directions, and the electron-hole pairs on different sides of the heterojunction are effectively separated in space before recombination.

4、Bi₂S₃ITO and NSCQDs/Bi₂S₃Characterization of ITO modified electrodes

Bi₂S₃ITO electrode and NSCQD/Bi₂S₃Open Circuit Potential (OCP) test of ITO electrodes as shown in fig. 9, the potential of the electrode was determined by redox balance in the absence of light (I region); when the illumination is turned on, the optical active material generates a large amount of photo-generated electrons to gatherThe electrode surface is concentrated, so that the OCP of the electrode is changed (area II); under the condition of continuous illumination, the photogenerated electron-hole pairs in the photoactive material can be recombined to a certain degree, so that the change of the OCP becomes smooth, and the OCP reaches a certain stable state (III region); when the light is turned off, the OCP gradually returns to the initial state (region IV) due to the recombination of a large number of photogenerated electron-hole pairs in the photoactive material and the electron donor in solution. As can be seen from region II in FIG. 9, when the light is turned on initially, NSCQD/Bi₂S₃Electrode ratio Bi of ITO (curve b)₂S₃The OCP of the/ITO electrode (curve a) changed more rapidly and to a greater extent, indicating NSCQD/Bi₂S₃The ITO electrode has higher photoproduction electron-hole separation speed and more electron enrichment amount on the surface of the electrode, which is attributed to the good optical property and electron transfer capability of NSCQDs. As can be seen from region III of FIG. 9, NSCQD/Bi after a period of illumination₂S₃ITO (curve b) electrode and Bi₂S₃The OCP of the/ITO electrodes (curve a) all reached a relatively steady state, while NSCQD/Bi₂S₃Ratio Bi of COP Change degree of/ITO (curve b) electrode₂S₃the/ITO electrode (curve a) is larger, which indicates that NSCQD/Bi2S3/ITO has a stronger optical response.

5. Characterization of photoelectrochemical bioimmune sensor construction Process

The modification process of the counter electrode was characterized using electrochemical impedance.

It can be seen from fig. 10(a) that the bare ITO electrode (curve a) exhibits a small semicircle. When Bi is compared with a bare ITO electrode₂S₃After the nano-rod is modified on the ITO electrode, Bi is added₂S₃The Ret value of the/ITO electrode (curve b) becomes smaller, which indicates that Bi is fixed to the electrode surface₂S₃The nanorods facilitate the transfer of electrons. NSCQDs/Bi after continuing to modify NSCQDs on the electrode surface₂S₃The Ret value of the/ITO electrode (curve c) is further reduced, which is attributed to the good conductivity of NSCQDs, further increasing the electron transfer rate. Ab is modified atAb/NSCQDs/Bi after the above-mentioned electrode₂S₃the/ITO electrode (curve d) shows a large Ret value, which is due to the insulating properties of the Ab. After the modification of BSA and the immunological recognition of AFP on the electrode surface is continued, the Ret values of the curve e and the curve f gradually increase because the electron transfer on the electrode surface is blocked step by step as the insulating protein is modified step by step on the electrode surface. The Ret value change of the electrode modified in different steps shows that the photoelectrochemistry biological immunosensor is successfully prepared.

For NSCQD/Bi under the conditions of illumination and no illumination respectively₂S₃ITO electrode and Bi₂S₃The impedance of the/ITO electrodes was tested and, as shown in FIG. 14, NSCQD/Bi at the illuminated bar entry compared to the non-illuminated condition₂S₃ITO electrode (curve b') and Bi₂S₃the/ITO electrodes (curve c') all show a smaller resistance value, which is attributed to the photoelectric effect of the photoactive material.

Cyclic Voltammetry (CV) was used to study the modification process of the photoelectrochemical biosensor. As shown in fig. 10 (B), an aligned reversible redox peak was obtained on the bare ITO electrode (curve a). When Bi is modified on the ITO₂S₃Then due to Bi₂S₃Has good conductivity, so that the oxidation peak current (curve b) becomes large. However, compared to Bi₂S₃ITO electrode, modified NSCQD/Bi₂S₃The oxidation peak current of the/ITO electrode (curve c) was further increased, indicating that NCQDs with good conductivity are effective in promoting electron transfer. In NSCQD/Bi₂S₃After the Ab, BSA and immuno-recognition AFP were sequentially modified on the ITO electrode, it can be seen that the oxidation peak current of the corresponding modified electrode (curve d-F) continuously decreased due to the weak conductivity and steric hindrance effect of Ab, BSA and AFP. Therefore, the photoelectrochemical biological immunosensor is successfully constructed as can be seen from the impedance change of the EIS curve and the change of the oxidation peak current of the CV curve.

Meanwhile, testing photocurrent signals of the electrodes modified in different steps. As shown in FIG. 10(C), 0 is contained.The bare ITO (curve a) electrode, tested in 15M AA in PBS (0.1M, pH 7.4) solution, had no significant photocurrent response. When Bi is present₂S₃After the nano-rod is modified on the ITO electrode, a stronger photocurrent signal is shown, because Bi₂S₃The nanorod has good photoelectric activity, and due to the existence of the electron donor AA, the separation efficiency of the photo-generated electron-hole pair is improved, so that a stronger photocurrent signal (curve b) is output. After the surface of the electrode is modified with the NSCQDs, the conductivity of the NSCQDs/Bi2S3/ITO electrode (curve c) and the utilization rate of visible light are improved, so that the photocurrent signal is further increased. After Ab and BSA are sequentially modified on the surface of the electrode and AFP is recognized through immunity, the electrical conductivity of the surface of the electrode is reduced due to the insulating property of protein molecules, and the transmission of AA to the surface of the electrode is hindered due to the steric hindrance effect of the protein molecules, so that the separation efficiency of photo-generated electron-hole pairs on the surface of the electrode is reduced, and Ab/NSCQDs/Bi₂S₃ITO electrode (curve d), BSA/Ab/NSCQDs/Bi₂S₃ITO electrode (curve d) and AFP/BSA/Ab/NSCQDs/Bi₂S₃The photocurrent signals of the/ITO (curve e) decrease in succession. The photoelectric signal change of the electrode obtained by continuous modification can be known, and the photoelectric chemical biological immunosensor is successfully prepared.

Example 2

NSCQDs/Bi prepared by the method₂S₃The photoelectrochemical sensor of (1) is used for the detection of ATP, and mainly comprises: 8 mu L of AFP solution with different concentrations is dripped in BSA/Ab/NSCQDs/Bi₂S₃The ITO electrode surface was incubated at room temperature for 1h, and then the electrode was washed with a PBS (0.1M, pH 7.4) solution. The obtained immunosensing electrode was used as a working electrode, an Ag/AgCl electrode as a reference electrode, and a platinum wire electrode as a counter electrode, and a photocurrent signal was measured in a solution containing 0.15M AA in PBS (0.1M, pH 7.4). Under the condition of no bias voltage, an optical filter (lambda is more than or equal to 420nm) is adopted to filter exciting light, and the illumination intensity is 20mW cm^-2And light irradiation/light blocking is carried out at intervals of 10s, and a photocurrent test is carried out.

Different concentrations of AFP (a-h)0.001, 0.01, 0.1, 1.0, 10, 100，300，500ng·mL^-1As shown in FIGS. 11(A) and (B), when the concentration of AFP is increased from 0.001 ng/mL-1 to 500 ng/mL-1, the steric hindrance of the electrode surface becomes large after the antigen-antibody pair is immunologically recognized, which hinders the approach of the electron sacrificial agent AA to the electrode surface, so that the separation rate of the photo-generated electron-hole is reduced, and the photocurrent signal of the AFP/BSA/Ab/NSCQDs/Bi2S3/ITO electrode gradually decreases. As can be seen from the inset in FIG. 11(B), the logarithm of the AFP concentration exhibits a linear dependence on the sensor photocurrent signal, which is given by the linear equation: i (μ a) ═ 2.076-0.1282logC (ng mL)^-1)(R₂0.9967), the detection limit was 1.1pg · mL-1(S/N — 3). Compared with the reported detection method, as shown in Table 1, the method for detecting AFP reported by the invention has lower detection limit and wider detection range, and the cutoff value of AFP in diagnosis is 25 ng-mL^-1Thus, the constructed photoelectrochemical biosensor enables sensitive analysis of AFP.

Table 1 comparison of the photoelectrochemical biosensor prepared according to the present invention with other methods for detection of AFP.

Under the same experimental conditions, 5 prepared sensing electrodes were used for 1ng · mL^-1The AFP of (1) was measured, and the measurement result is shown in FIG. 15(A), and the relative standard deviation of the photocurrent signals of 5 sensors is 1.32%, which indicates that the prepared sensor has good reproducibility. Meanwhile, the prepared sensing electrode was stored in a refrigerator at 4 ℃ for various periods of time, and then the temperature was adjusted to 0.1 ng/mL^-1The AFP of (1) was measured, and the results are shown in FIG. 15(B), and when the storage time was 2 weeks, the photocurrent signal obtained by the measurement was 92.4% of the initial signal, which indicates that the prepared sensor had good stability. In order to examine the AFP specificity detection of the prepared sensor, the prepared sensor was applied to a blank solution and 10 ng-mL^-1AFP, 100 ng.mL^-1CEA, 100 ng. mL^-1PSA, 100n ofg·mL^-1The BSA interfering substance and the mixture thereof were tested, and the results are shown in fig. 15(C), when AFP solution and the mixture thereof are used, the photocurrent signal of the sensor is significantly reduced due to the specific immunological binding of Ab to AFP. The above results indicate that the prepared sensor has excellent selectivity.

To investigate the detection side ability of the sensor to the actual sample, a recovery test was performed by applying standard additive methods to human serum samples. Serum samples were first centrifuged at 8000r/min for 15min, the supernatant removed and diluted 10-fold with PBS (0.01M, pH 7.4). AFP solutions of various known concentrations were then added to the above serum samples for testing. The experimental results are shown in table 2, the tested recovery rate is between 95.0-103.7%, and the relative standard deviation is in the range of 2.17-3.31%, which indicates that the constructed sensor has potential clinical application prospect for AFP in human serum samples.

TABLE 2 recovery of AFP detection in human serum samples by photoelectrochemical biosensors

Example 3

In order to make the prepared photoelectrochemistry biological immunosensor show the most excellent performance, relevant parameters of the preparation process are optimized as follows.

In the construction process of the photoelectrochemical biosensor, Bi₂S₃The concentration of (a) plays a very important role in the performance of the sensor. As shown in FIG. 12(A), with Bi₂S₃Concentration of (2) is from 0.5 mg.mL^-1Increased to 3.0 mg. mL^-1，Bi₂S₃The photocurrent of the/ITO electrode gradually increases; continuously increasing Bi₂S₃The concentration is 7 mg/mL^-1When is Bi₂S₃The photocurrent of the/ITO electrode starts to gradually decrease. This is because of the following Bi₂S₃Increase in concentration of (3), Bi formed on the surface of the electrode₂S₃The layer will changeThick, which not only hinders electron transport but also increases the recombination probability of photo-generated electron-hole pairs, thereby causing a decrease in the output photocurrent signal. Therefore, 3.0 mg/mL was selected^-1Of Bi₂S₃The subsequent experiments were continued.

As an electron sacrificial agent, AA can improve the separation efficiency of photogenerated electron-hole pairs, thereby optimizing the concentration of AA in the solution. As shown in FIG. 12(B), the concentration of AA was in the range of 0.01 to 0.3M for Bi₂S₃The photoelectricity of the/ITO electrodes was tested. When the concentration of AA increased to 0.15M, Bi₂S₃The photoelectric signal of the ITO electrode is maximum; as the AA concentration continued to increase, Bi was found₂S₃The photoelectric signal of the/ITO electrode tends to decline. This is because AA in a low concentration range can effectively promote the separation of photo-generated electron-hole pairs, enhancing the photocurrent signal; while a high concentration of AA will increase the absorption of light by the solution, resulting in a decrease in the light intensity impinging on the electrode surface and a decrease in the photocurrent signal. Therefore, 0.15M AA was selected for subsequent experiments.

FIG. 12(C) shows the different concentrations of NSCQDs vs. NSCQDs/Bi₂S₃Photocurrent influence of/ITO electrodes. With the concentration from 0.5 mg/mL^-1Increased to 2.0 mg. mL^-1，NSCQDs/Bi₂S₃The photocurrent of the/ITO electrode shows a rising trend; when the concentration is continuously increased to 3.5 mg. mL^-1In time, NSCQDs/Bi₂S₃The photocurrent of the/ITO electrode gradually decreased. When the low-concentration NSCQDs are used for modifying the electrode, the NSCQDs modified on the surface of the electrode have good conductivity and up-conversion fluorescence property, so that the utilization rate of light can be improved by the modified electrode pair, and a photocurrent signal is improved; when the concentration of the NSCQDs is too high, the NSCQDs modified on the surface of the electrode are agglomerated, which is not only unfavorable for the transmission of electrons, but also hinders Bi₂S₃Absorption of visible light results in a drop in the output photocurrent signal. It can be seen that the concentration of NSCQDs is 2.0 mg. mL^-1Time NSCQDs/Bi₂S₃The ITO electrode showed the largest photoelectric signal, so that NSC of 2.0 mg. mL-1 was selectedQDs were subjected to the next step.

At the same time, the pH value of the solution is different for NSCQDs/Bi₂S₃The performance of the/ITO electrodes was tested. As can be seen from FIG. 12(D), when the pH of the solution was varied within the range of 5.6 to 8.6, NSCQDs/Bi at a solution pH of 7.4₂S₃The photocurrent signal of the/ITO electrode was optimal and the pH of 7.4 was chosen for the next experiment, taking into account the subsequent Ab modification on the electrode.

The modification amount of Ab on the surface of the electrode directly determines the detection performance of the sensing electrode on AFP. As shown in FIG. 13 (A), the Ab concentration on the electrode surface was adjusted from 5. mu.g.mL with the dropwise addition^-1Increased to 30. mu.g.mL^-1Ab/NSCQDs/Bi₂S₃The photocurrent of the ITO electrode gradually decreases; when the Ab concentration is from 30. mu.g.mL^-1Further increase to 50. mu.g/mL^-1There was little apparent change in photocurrent of the modified electrode, indicating that Ab immobilization on the electrode surface tended to saturate. Therefore, 30. mu.g/mL was selected^-1Ab (c) to modify the electrode.

Similarly, the incubation time of abs on the electrode surface was also studied. As can be seen from fig. 13 (B). When the hatching time is prolonged from 2h to 6h, Ab/NSCQDs/Bi₂S₃The photoelectricity of the/ITO electrode showed a tendency to decrease, indicating that the amount of Ab immobilized on the electrode surface gradually increased with the increase of the incubation time; when the incubation time is continued to be prolonged to 12h, the photocurrent signal of the modified electrode is obviously changed. This indicates that 6h incubation time was sufficient to allow the Ab to firmly decorate the electrode surface. Thus 6h incubation was chosen for sensor construction.

The relationship between the immunoreaction time of Ab and AFP and the photocurrent signal of the sensor was explored, as shown in FIG. 13(C), with the increase in immunoreaction time from 20min to 60min, AFP/Ab/NSCQDs/Bi₂S₃The photocurrent signal of the/ITO electrode always decreased. When the immunization time is continued to be prolonged to 120min, the photoelectric signal of the sensor has no obvious change, which indicates that the immune reaction is basically completed at 60 min. Therefore, the subsequent experiment was performed with an immunization time of 60 min.

Claims

1. Based on NSCQDs/Bi₂S₃The preparation method of the photoelectrochemical sensor is characterized by mainly comprising the following steps:

(1) preparation of Bi₂S₃A nanorod;

(2) synthesizing NSCQDs;

(3) adding Bi₂S₃Adding the nano-rod into deionized water, and performing ultrasonic dispersion to obtain Bi₂S₃Dripping the suspension on an ITO conductive substrate, drying in an oven at 60 ℃ for 20-40min, taking out, and naturally cooling to room temperature to obtain Bi₂S₃An ITO modified electrode;

(4) bi obtained in step (3)₂S₃Dropwise adding a PDDA solution containing NaCl on the surface of the ITO modified electrode, standing at room temperature for 10-20min, cleaning the electrode with deionized water, and then drying with nitrogen;

(6) the NSCQDs/Bi obtained in the step (5) are mixed₂S₃Soaking the ITO modified electrode into a solution containing EDC and NHS, activating for 1h at room temperature, and washing the electrode by using a PBS solution to wash away redundant EDC and NHS;

(7) dropping AFP antibody solution in NSCQDs/Bi₂S₃ITO surface, and placing the ITO surface in a refrigerator at 4 ℃ for incubation for 6 h; after incubation, washing the electrodes with PBS solution to remove the physically adsorbed AFP antibodies; then, adding BSA solution dropwise to the surface of the electrode, placing the electrode in a refrigerator at 4 ℃, and placing for 2 hours to block off the active sites of non-specific binding; then washed with PBS solution.

2. The NSCQDs/Bi-based device of claim 1₂S₃The method for preparing a photoelectrochemical sensor of (1), wherein the Bi is in the step (1)₂S₃The preparation of the nano-rod mainly comprises the following steps: weighing Bi (NO)₃)·5H₂The O is dissolved in the ethylene glycol,stirring to form a transparent solution A; then weighing Na₂S·9H₂Placing the O in ethylene glycol and deionized water, and stirring to form a transparent solution B; adding the solution B into the solution A dropwise under the condition of strong stirring; then adding urea and deionized water into the solution, stirring strongly, transferring the solution into a reaction kettle, and placing the reaction kettle at 180 ℃ for reaction for 24 hours; after the reaction is finished, naturally cooling to room temperature, washing the obtained black product with water and alcohol, and finally placing the black product in an oven at 60 ℃ for 8 hours.

3. The NSCQDs/Bi-based device of claim 1₂S₃The method for preparing the photoelectrochemical sensor is characterized in that the synthesis of the NSCQDs in the step (2) mainly comprises the following steps: weighing ammonium citrate and thiourea, placing the ammonium citrate and the thiourea in deionized water, violently stirring to dissolve the ammonium citrate and the thiourea, transferring the ammonium citrate and the thiourea into a reaction kettle, placing the reaction kettle in an oven, and reacting for 4 hours at 160 ℃; after the reaction is finished, naturally cooling to room temperature, centrifuging the obtained solution to remove precipitates, and then transferring the solution to a dialysis bag for dialysis for 48 hours; followed by rotary evaporation and freeze drying.

4. The NSCQDs/Bi-based device of claim 1₂S₃The method for producing a photoelectrochemical sensor of (1), wherein said Bi in the step (3)₂S₃The concentration of the suspension was 3.0 mg/mL^-1。

5. The NSCQDs/Bi-based device of claim 1₂S₃The method for preparing a photoelectrochemical sensor of (1), wherein the concentration of the NSCQDs solution in the step (5) is 2.0 mg/mL^-1。

6. The NSCQDs/Bi-based device of claim 1₂S₃Characterized in that the concentration of the AFP antibody solution in the step (7) is 30. mu.g.mL^-1。

7. The NSCQDs/Bi-based device of claim 1₂S₃The method for producing a photoelectrochemical sensor according to (1), wherein the concentration of PBS is 0.1M and the pH is 7.4.

8. NSCQDs/Bi-based prepared by the preparation method according to claim 1₂S₃The photoelectrochemical sensor of (1).