CN114088786A - Construction method of visualized electrochemiluminescence sensor based on ruthenium (II) complex and application of visualized electrochemiluminescence sensor in detection of OTA - Google Patents
Construction method of visualized electrochemiluminescence sensor based on ruthenium (II) complex and application of visualized electrochemiluminescence sensor in detection of OTA Download PDFInfo
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- YAYGSLOSTXKUBW-UHFFFAOYSA-N ruthenium(2+) Chemical compound [Ru+2] YAYGSLOSTXKUBW-UHFFFAOYSA-N 0.000 title claims abstract description 7
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- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 claims abstract description 48
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 claims abstract description 43
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Abstract
The invention provides a method for constructing a visual electrochemical luminescence sensor based on a ruthenium (II) complex, which utilizes BiOI to Ru (bpy)3 2+The fixed reinforcing effect of (2) need not to adopt traditional Photomultiplier (PMT) to enlarge the tactics, designs two regions on ITO, realizes separating visualization unit and detecting element, and the visual detection of electrochemiluminescence based on ruthenium (II) complex luminous body luminous intensity changes, the step is as follows: step 1, preparing luminescent material bipyridine ruthenium-iodine-oxygen bismuth composite microspheres (Ru (bpy)3 2+a/BiOI); and 2, constructing a visual electrochemiluminescence aptamer sensor for detecting ochratoxin A. Based on thisThe portable detection mode integrates the advantages of rapidness, simplicity, miniaturization, flexible use mode and the like, and can realize on-site rapid detection.
Description
Technical Field
The invention belongs to the technical field of electrochemiluminescence biosensing, and mainly relates to a construction method of a novel ruthenium (II) complex-based visual electrochemiluminescence sensor and application of the sensor in detection of ochratoxin A.
Background
Electrochemiluminescence, or Electrochemiluminescence (ECL), is the process of producing light radiation by chemical reaction between products of electrode reactions or between an electrode product and a component in a system, and is the product of combining chemiluminescence with electrochemistry. It not only retains the advantages of chemiluminescence method, but also has many advantages incomparable with chemiluminescence method.
An electrochemiluminescence sensor is a type of experimental device constructed by directly or indirectly fixing a reagent participating in a chemiluminescence reaction on a working electrode by a chemical modification method, and is generally called as an electrochemiluminescence sensor or an electrochemiluminescence sensor. The electrochemical luminescence sensor not only retains the advantage of electrochemical luminescence, but also overcomes the defect that the popularization and the application of the electrochemical luminescence analysis method are limited due to poor selectivity and the use of a complex optical system and a solution type electrochemical luminescence reagent in the traditional electrochemical luminescence analysis, widens the application range of the electrochemical luminescence analysis method, realizes the miniaturization of an instrument and increases the practicability of the method. However, visualization of electrochemiluminescence imaging ECL signal visualization sensing is more suitable for in situ detection of mycotoxins than conventional ECL bioanalytical systems using photomultiplier tubes (PMT) without the need for special instrumentation. The visual electrochemical luminescence sensor has the advantages of simple operation steps, direct visual observation of detection results, no need of an external instrument for detection, easiness in portability and the like, and has wide application in the field of environment detection and food detection.
Ochratoxin a (ota) is a secondary metabolite produced by a number of aspergillus and penicillium fungi. OTA has stable physicochemical properties, is widely present in cereal-related products such as corn, wheat and oat, and attracts wide attention due to high toxicity. The ingestion of OTA toxins is a great threat to both humans and animals. Mainly damaging hematopoietic organs, immune organs, digestive system, nervous system and reproductive system. In view of the hazardousness of OTA, it has been determined by the food and agriculture organization and the world health organization of the united nations to be one of the most dangerous naturally occurring food contaminants. At present, the detection methods for OTA include enzyme-linked immunosorbent assay, electrochemical immunosensing detection, biosensor detection, and the like. The detection method has the advantages of single detection mode, complex operation, inconvenience for carrying and incapability of realizing field rapid detection. Therefore, it is necessary to develop an analysis method with flexible detection mode, high speed, simplicity, portability and low cost.
Disclosure of Invention
The invention aims to provide a portable visual electrochemical luminescence aptamer sensor which integrates the advantages of rapidness, simplicity, miniaturization, flexible use mode and the like and is applied to the detection of OTA toxin, and the BiOI is utilized to Ru (bpy)3 2+The fixed enhancement effect of (2) need not to adopt traditional Photomultiplier (PMT) magnification strategy, designs two regions on ITO, realizes separating visualization unit and detecting element, the visual detection of electrochemiluminescence based on ruthenium (II) complex luminous body luminous intensity change.
The construction method of the visual electrochemical luminescence sensing device comprises the following steps:
step 1, preparing luminescent material bipyridine ruthenium-iodine-oxygen bismuth composite microspheres (Ru (bpy)3 2+/BiOI)：
Firstly, adding bipyridyl ruthenium into bismuth oxyiodide dispersion liquid, and stirring to form uniformly dispersed aqueous solution A; then, the aqueous solution A was transferred to an ultrasonic machine and subjected to ultrasonication, whereby Ru (bpy) was successfully produced3 2+a/BiOI microsphere composite material; finally, by washing with water and ethanol several times, drying was carried out to prepare Ru (bpy) as a solid product3 2+a/BiOI microsphere.
Performing ultrasonic cleaning on ITO in toluene, acetone, ethanol and water in sequence, drying in nitrogen flow, performing electrochemical cleaning on ITO glass by adopting a cyclic voltammetry method to remove impurities and organic pollutants which can be adsorbed on the surface, and finally drying by using nitrogen; a visual area and a detection area are etched on the clean ITO through laser;
step 3, modifying the visual area
The solid product obtained in step 1, Ru (bpy)3 2+the/BiOI is dispersed in deionized water to obtain Ru (bpy)3 2+a/BiOI dispersion of Ru (bpy)3 2+the/BiOI dispersion liquid is dripped on a visual area of ITO, Nafion solution is dripped to form a uniform film fixing material, and the uniform film fixing material is placed in an oven to be dried to obtain a visual electrode Ru (bpy)3 2+a/BiOI/Nafion electrode.
Step 4, modifying the detection area:
firstly, spin-coating a Graphene Oxide (GO) solution in a detection area, and standing at room temperature to form a transparent film;
then, PBS is used as a solvent to prepare an OTA aptamer solution, the OTA aptamer solution is dripped on a transparent film of graphene oxide, after incubation for 10 hours at 4 ℃, PBS is used for leaching to remove any unbound aptamer, and then Bovine Serum Albumin (BSA) solution is dripped to seal the nonspecific active site; finally obtaining aptamer/GO/ITO modified detection area;
step 5, constructing a visual electrochemiluminescence aptamer sensor for detecting OTA toxin
Step 4, obtaining aptamer modified detection region aptamer/GO/ITO and step 3, wherein the visualization region Ru (bpy)3 2+the/BiOI/Nafion forms a visual electrochemical luminescence aptamer sensing device.
In the step 1, in the solution A, the proportion of the used amount of the bipyridyl ruthenium, the bismuth oxyiodide and the deionized water is 0.375 g: 0.042 g: 50 mL; the drying temperature is 60 ℃, and the drying time is 12 h.
In the step 2, the time of ultrasonic treatment is 15min, the parameters of cyclic voltammetry are set to scan for 15 times within the range of 0-1.5V in 0.1mol/L sodium hydroxide solution, then scan for 30 times within the range of 0-1.6V in 0.5mol/L sulfuric acid solution, the scanning speed is 100mV/s, and the time for introducing high-purity nitrogen (99.999%) is 10 min.
In step 3, Ru (bpy)3 2+The concentration of the/BiOI dispersion liquid is 2mg/mL, and the dripping amount is 20 mu L; the mass percentage concentration of the Nafion solution is 0.5 percent, and the dropping amount is 10 mu L; the stemThe drying temperature is 60 ℃ and the drying time is 2 h.
In the step 4, the mass percentage concentration of the Graphene Oxide (GO) solution is 0.2%, the dropping amount is 20 mu L, and the standing time is 2 h.
In the step 4, the process of the method,
the concentration of the PBS solution is 0.1 mol/L;
the OTA aptamer sequence is: 5'-GATC GGGT GTGG GTGG CGTA AAGG GAGC ATCG GACA A-3', respectively; the concentration of the OTA aptamer is 4 mu M, the dropping amount is 20 mu L, and the incubation time is 10 h; the BSA concentration was 3% by mass and the amount added was 20. mu.L.
The application of the visual electrochemiluminescence aptamer sensor prepared by the invention in detection of ochratoxin OTA comprises the following specific steps:
(1) dripping OTA solution with different concentrations to a detection area on an aptamer/GO/ITO electrode, and incubating for a period of time at room temperature;
(2) putting the electrode treated in the step (1) into PBS electrolyte containing butyl diethanol amine (DBAE), connecting the electrode with an electrochemical workstation by using a conductive adhesive tape, and scanning the electrochemical workstation at a constant potential by using Ru (bpy)3 2+the/BiOI luminous intensity is used as an output signal; ru (bpy)3 2+Making a standard curve by the change of RGB value and OTA concentration during the luminescence of the/BiOI;
(3) and collecting signals of the OTA solution with unknown concentration by adopting the method, and substituting the signals into the standard curve to obtain the concentration of the OTA solution.
In the step (1), the concentration of the OTA is 1-100 ng/mL, specifically 1,10,25,50,75 and 100ng/mL, and the dropping amount is 20 mu L;
in the step (2), the concentration of DBAE is 20 mM; the amount of PBS is 20-30 mL; the constant potential is 1.3V, and the reaction time is 30 s.
The invention has the beneficial effects that:
the invention takes ITO with functional area as a substrate to modify Ru (bpy)3 2+The area of the/BiOI microsphere is used as a visual area, the modified Graphene Oxide (GO) area is used as a detection area, the visual electrochemiluminescence aptamer sensor is successfully established, and the OTA toxin is fixedQuantitative analysis and detection, its characteristics and advantages are expressed as follows:
(1) the invention prepares Ru (bpy)3 2+the/BiOI microspheres are used as a visual area luminophor, the visual electrochemical luminescence aptamer sensor is constructed by using the modified graphene oxide substrate as a detection area, and the luminescence signal is stable.
(2) The invention prepares Ru (bpy)3 2+Enhanced Ru (bpy) by/BiOI microspheres3 2+The OTA toxin can be visually detected based on the electrode disclosed by the invention, an electrochemical luminescence detection instrument is not needed, the detection complexity is reduced, and the cost is reduced.
(3) The visual electrochemiluminescence aptamer sensor provided by the invention realizes the sensitive detection of OTA, and the concentration of the OTA and Ru (bpy) are within the concentration range of 1-100 ng/mL3 2+The change of RGB values during the emission of the/BiOI presents a good linear relation, and the detection limit can reach 0.33 ng/mL.
(4) The visual electrochemiluminescence aptamer sensor constructed by the invention does not need to rely on the traditional connection mode of a photomultiplier tube (PMT) for detection. The electrode based on the invention can realize outdoor portable luminescence detection, can also realize high-sensitivity quantitative detection, achieves the purpose of timely detection, and has flexible use mode.
Drawings
FIG. 1 is a schematic diagram of a constructed visual electrochemiluminescent aptamer sensor;
FIG. 2 is a schematic diagram of an ITO electrode area of the present invention;
FIG. 3 shows Ru (bpy) for preparation3 2+A scanning electron microscope picture (A) and an X-ray diffraction picture (B) of the/BiOI microspheres; an X-ray photoelectron spectrum (C);
FIGS. 4(A) and (B) show the OTA concentration and Ru (bpy)3 2+An RGB value change relation graph when the/BiOI microspheres emit light, and a result graph of sensor selectivity test.
Detailed Description
The present invention is described in detail below with reference to the drawings and examples of the specification, but the present invention is not limited to these embodiments.
FIG. 1 is a mechanism diagram of detection of a constructed visual electrochemiluminescence aptamer sensor, and FIG. 2 is a schematic diagram of an ITO electrode area of the invention.
(1)Ru(bpy)3 2+Preparation of/BiOI microspheres
1.455g of bismuth nitrate pentahydrate and 0.492g of potassium iodide were dissolved in a beaker containing 20mL of an ethylene glycol solution, respectively, and stirred to form a homogeneous solution. Then, the bismuth nitrate solution was added dropwise to the potassium iodide solution, with vigorous stirring, and the solution gradually changed from an initially clear solution to an orange suspension. And transferring the stirred suspension into a 50mL polytetrafluoroethylene autoclave, reacting for 12h at the temperature of 140 ℃, respectively washing the sample for 3 times by using ethanol and deionized water after the autoclave is naturally cooled to the room temperature, and finally drying for 12h in a 60 ℃ drying oven to obtain the BiOI microspheres.
Different amounts of Ru (bpy)3 2+And BiOI are dispersed in water, ultrasonic treatment is carried out for 2h, stirring and mixing are carried out for 6h, and finally drying is carried out at 60 ℃ to obtain Ru (bpy)3 2+/BiOI microsphere particles.
The characterization results are shown in FIG. 3, and the successful preparation of BiOI microspheroidal particles can be seen from SEM and XRD in FIGS. A and B, for Ru (bpy) in FIG. C3 2+XPS (X Performance ray diffraction) tests of the/BiOI microsphere particles analyze the element composition of the/BiOI microsphere particles and successfully prove the preparation of the composite material.
(2) Electrode substrate pretreatment
The electrode of the invention is mainly completed in two parts, including the manufacture of the base layer and the manufacture of the reaction region. First, ITO glass having a stripe pattern is formed by pattern etching. In brief, the ITO surface was first cleaned sequentially with acetone, ethanol and deionized water, and nitrogen blown dry for use. And then electrochemically cleaning the ITO glass by adopting a cyclic voltammetry method to remove impurities and organic pollutants which can be adsorbed on the surface, and displaying a pattern with a functional area on the clean ITO by using laser etching. Independent circular detection units (r is 8mm) and visualization units (r is 8mm) are formed on the surface of the ITO glass.
(3) Modified visualization electrode
Using 8mm round brown colour to resist high tempFixing the working electrode area with adhesive tape, weighing 2mg Ru (bpy)3 2+the/BiOI was dispersed in 1mL deionized water to give Ru (bpy)3 2+the/BiOI dispersion was removed 20. mu.L of Ru (bpy)3 2+Uniformly dripping/coating the/BiOI dispersion liquid on a visible electrode area, continuously dripping 10 mu L of Nafion solution with the mass concentration of 0.5%, and drying in an oven at 60 ℃ for 2h to obtain Ru (bpy)3 2+/BiOI/Nafion。
(4) Construction of visual electrochemiluminescence aptamer sensor
And spin-coating 20 mu L of Graphene Oxide (GO) solution on the electrode detection area, wherein the mass percentage concentration of the Graphene Oxide (GO) solution is 0.2%, and the standing time is 2 h. A 4 μ M OTA aptamer solution was prepared in PBS (pH 7.4, 0.1mol/L) as solvent with the sequence: 5'-GATC GGGT GTGG GTGG CGTA AAGG GAGC ATCG GACA A-3' are provided. Dropping 20 μ L of OTA aptamer on the electrode detection area, reacting in a refrigerator at 4 deg.C for 10h, rinsing with PBS to remove excessive unadsorbed aptamer, dropping 20 μ L of 3% Bovine Serum Albumin (BSA) solution to block nonspecific active site,
the final aptamer modified detection region (aptamer/GO/ITO) and visualization region (Ru (bpy))3 2+/BiOI/Nafion) to form a visual electrochemiluminescence aptamer sensing device
(5) Visual electrochemiluminescence aptamer sensor detection OTA
mu.L of OTA toxin at concentrations of 1,10,25,50,75 and 100ng/mL were dropped onto the detection area of the electrodes, respectively, and incubated at room temperature for 40 min. Finally, the electrode was placed in an electrolytic cell of PBS (pH 7.4, 0.1mol/L) at a concentration of 20mM butyldiethanolamine (DBAE), the amount of the solution being 30 mL. And connecting the electrode with an electrochemical workstation by using a conductive adhesive tape, and carrying out constant potential scanning by the electrochemical workstation, wherein the potential is 1.3V and the reaction time is 30 s. With Ru (bpy)3 2+the/BiOI luminous intensity is used as an output signal; ru (bpy)3 2+The change of RGB value during the luminescence of/BiOI is chemically analyzed.
The detection results are shown in fig. 4, and fig. 4 shows the detection results of the sensor in the visualization mode under different OTA concentrations. As can be seen from the graph A, with the increase of the concentration of OTA, Ru (bpy)3 2+The larger the amount of color of the/BiOI luminescence, the larger the change in RGB value. As shown in the graph B, the concentration of OTA and Ru (bpy) was found in the concentration range of 1-100 ng/mL3 2+The change of RGB values during the emission of the/BiOI presents a good linear relation, the detection limit can reach 0.33ng/mL, and a graph C shows that the sensor has good selectivity.
Claims (9)
1. The construction method of the visual electrochemical luminescence sensor based on the ruthenium (II) complex is characterized by comprising the following steps:
step 1, preparing luminescent material bipyridine ruthenium-iodine-oxygen bismuth composite microspheres Ru (bpy)3 2+/BiOI：
Firstly, adding bipyridyl ruthenium into bismuth oxyiodide dispersion liquid, and stirring to form uniformly dispersed aqueous solution A; then, the aqueous solution A was transferred to an ultrasonic machine and subjected to ultrasonication, whereby Ru (bpy) was successfully produced3 2+a/BiOI microsphere composite material; finally, it was washed and dried to prepare Ru (bpy) as a solid product3 2+a/BiOI microsphere;
step 2, preparing a visual electrode substrate
Performing ultrasonic cleaning on ITO in toluene, acetone, ethanol and water in sequence, drying in nitrogen flow, performing electrochemical cleaning on ITO glass by adopting a cyclic voltammetry method to remove impurities and organic pollutants which can be adsorbed on the surface, and finally drying by using nitrogen; a visual area and a detection area are etched on the clean ITO through laser;
step 3, modifying the visual area
The solid product obtained in step 1, Ru (bpy)3 2+the/BiOI is dispersed in deionized water to obtain Ru (bpy)3 2+a/BiOI dispersion of Ru (bpy)3 2+the/BiOI dispersion liquid is dripped on a visual area of ITO, Nafion solution is dripped to form a uniform film fixing material, and the uniform film fixing material is placed in an oven to be dried to obtain a visual electrode Ru (bpy)3 2+a/BiOI/Nafion electrode;
step 4, modifying the detection area:
firstly, spin-coating a graphene oxide GO solution in a detection area, and standing at room temperature to form a transparent film;
then, PBS is used as a solvent to prepare an OTA aptamer solution, the OTA aptamer solution is dripped on a transparent film of graphene oxide, after a certain period of incubation, PBS is used for leaching to remove any unbound aptamer, and then bovine serum albumin BSA solution is dripped to seal the nonspecific active site; finally obtaining aptamer/GO/ITO modified detection area;
step 5, constructing a visual electrochemiluminescence aptamer sensor for detecting OTA toxin
Step 4, obtaining aptamer modified detection region aptamer/GO/ITO and step 3, wherein the visualization region Ru (bpy)3 2+the/BiOI/Nafion forms a visual electrochemical luminescence aptamer sensing device.
2. The method according to claim 1, wherein in step 1, the ratio of the amount of bipyridyl ruthenium, bismuth oxyiodide and deionized water in solution A is 0.375 g: 0.042 g: 50 mL; the drying temperature is 60 ℃, and the drying time is 12 h.
3. The method of claim 1, wherein in step 2, the time of ultrasound is 15min, the parameters of cyclic voltammetry are set to scan 15 times in the range of 0-1.5V in 0.1mol/L sodium hydroxide solution, then scan 30 times in the range of 0-1.6V in 0.5mol/L sulfuric acid solution, the scan rate is 100mV/s, and the time of introducing high purity nitrogen (99.999%) is 10 min.
4. The method of claim 1, wherein in step 3, Ru (bpy)3 2+The concentration of the/BiOI dispersion liquid is 2mg/mL, and the dripping amount is 20 mu L; the mass percentage concentration of the Nafion solution is 0.5 percent, and the dropping amount is 10 mu L; the drying temperature is 60 ℃, and the drying time is 2 h.
5. The construction method according to claim 1, wherein in the step 4, the Graphene Oxide (GO) solution has a mass percentage concentration of 0.2%, a dropping amount of 20 μ L and a standing time of 2 h.
6. The method of construction according to claim 1,
in step 4, the concentration of the PBS solution is 0.1 mol/L;
the OTA aptamer sequence is: 5'-GATC GGGT GTGG GTGG CGTA AAGG GAGC ATCG GACA A-3', respectively; the concentration of the OTA aptamer is 4 mu M, and the dropping amount is 20 mu L; the incubation temperature is 4 ℃ and the time is 10 h; the BSA concentration was 3% by mass and the amount added was 20. mu.L.
7. Use of the visual electrochemiluminescence aptamer sensor constructed by the construction method of any one of claims 1-6 for detecting ochratoxin A.
8. The use according to claim 7, wherein the specific step of detection is
(1) Dripping OTA solution with different concentrations to a detection area on an aptamer/GO/ITO electrode, and incubating for a period of time at room temperature;
(2) putting the electrode treated in the step (1) into PBS electrolyte containing butyldiethanolamine DBAE, connecting the electrode with an electrochemical workstation by using a conductive adhesive tape, and carrying out constant potential scanning by the electrochemical workstation by using Ru (bpy)3 2+the/BiOI luminous intensity is used as an output signal; ru (bpy)3 2+Making a standard curve by the change of RGB value and OTA concentration during the luminescence of the/BiOI;
(3) and collecting signals of the OTA solution with unknown concentration by adopting the method, and substituting the signals into the standard curve to obtain the concentration of the OTA solution.
9. The use according to claim 8,
in the step (1), the concentration of the OTA is 1-100 ng/mL, and the dripping amount is 20 mu L;
in the step (2), the concentration of DBAE is 20 mM; the amount of PBS is 20-30 mL; the constant potential is 1.3V, and the reaction time is 30 s.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040175693A1 (en) * | 2003-03-07 | 2004-09-09 | Yi Lu | Nucleic acid biosensors |
| CN110501408A (en) * | 2019-07-09 | 2019-11-26 | 江苏大学 | A kind of Photoelectrochromic visual biosensor of sketch-based user interface principle and its preparation method and application |
| US20200300849A1 (en) * | 2016-03-25 | 2020-09-24 | Kansas State University Research Foundation | Nanosensors and methods for detection of biological markers |
| CN112666156A (en) * | 2020-12-17 | 2021-04-16 | 中南大学 | Method for visual specific detection of biomacromolecule based on effective assembly of aptamer and gold nanoparticles |
| CN113295741A (en) * | 2021-06-16 | 2021-08-24 | 江苏大学 | Method for constructing photoelectric photochromic visual sensor for simultaneously detecting multiple organic pollutants |
| CN113398994A (en) * | 2021-06-25 | 2021-09-17 | 西北大学 | Keggin type heteropoly acid insoluble salt heterojunction catalyst and preparation method and application thereof |
-
2021
- 2021-10-19 CN CN202111214436.7A patent/CN114088786B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040175693A1 (en) * | 2003-03-07 | 2004-09-09 | Yi Lu | Nucleic acid biosensors |
| US20200300849A1 (en) * | 2016-03-25 | 2020-09-24 | Kansas State University Research Foundation | Nanosensors and methods for detection of biological markers |
| CN110501408A (en) * | 2019-07-09 | 2019-11-26 | 江苏大学 | A kind of Photoelectrochromic visual biosensor of sketch-based user interface principle and its preparation method and application |
| CN112666156A (en) * | 2020-12-17 | 2021-04-16 | 中南大学 | Method for visual specific detection of biomacromolecule based on effective assembly of aptamer and gold nanoparticles |
| CN113295741A (en) * | 2021-06-16 | 2021-08-24 | 江苏大学 | Method for constructing photoelectric photochromic visual sensor for simultaneously detecting multiple organic pollutants |
| CN113398994A (en) * | 2021-06-25 | 2021-09-17 | 西北大学 | Keggin type heteropoly acid insoluble salt heterojunction catalyst and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| QIAN ZHANG等: "Recent Advances in Visual Electrochemiluminescence Analysis" * |
| YUAN WANG等: "Visible light-driven photoelectrochemical ampicillin aptasensor based on an artificial Z-scheme constructed from Ru(bpy)3 2+-sensitized BiOI microspheres" * |
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