CN114088786B - Construction method of visualized electrochemical luminescence sensor based on ruthenium (II) complex and application of visualized electrochemical luminescence sensor in detection of OTA - Google Patents
Construction method of visualized electrochemical luminescence sensor based on ruthenium (II) complex and application of visualized electrochemical luminescence sensor in detection of OTA Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 53
- 238000004020 luminiscence type Methods 0.000 title claims abstract description 18
- 238000010276 construction Methods 0.000 title claims abstract description 10
- YAYGSLOSTXKUBW-UHFFFAOYSA-N ruthenium(2+) Chemical compound [Ru+2] YAYGSLOSTXKUBW-UHFFFAOYSA-N 0.000 title claims abstract description 7
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 claims abstract description 50
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- 230000008901 benefit Effects 0.000 abstract description 8
- 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 abstract description 3
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 abstract description 2
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- FBXVOTBTGXARNA-UHFFFAOYSA-N bismuth;trinitrate;pentahydrate Chemical compound O.O.O.O.O.[Bi+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O FBXVOTBTGXARNA-UHFFFAOYSA-N 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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Abstract
The invention provides a construction method of a visual electrochemical luminescence sensor based on ruthenium (II) complex, which utilizes BiOI to carry out Ru (bpy) 3 2+ The fixed enhancement effect of (2) does not need to adopt the traditional Photomultiplier (PMT) amplification strategy, two areas are designed on ITO, the separation of a visual unit and a detection unit is realized, and the electrochemical luminescence visual detection based on the change of the luminescence intensity of the ruthenium (II) complex luminophor is realized, wherein the steps are as follows: step 1, preparing a luminescent material bipyridine ruthenium-iodine bismuth oxide composite microsphere (Ru (bpy) 3 2+ BiOI); and 2, constructing a visual electrochemiluminescence aptamer sensing device for detecting ochratoxin A. Based on the portable detection mode, the 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 electrochemical luminescence biosensing, and mainly relates to a novel construction method of a visual electrochemical luminescence sensor based on ruthenium (II) complex and application of the visual electrochemical luminescence sensor in detection of ochratoxin A.
Background
Electrochemiluminescence, or Electrochemiluminescence (ECL), is the process of generating optical radiation by chemical reaction between electrode reaction products or between an electrode product and a component in a system, and is the product of combining chemiluminescence with electrochemistry. It retains the advantages of the chemiluminescent method, and has many incomparable advantages of the chemiluminescent method.
Electrochemical luminescence sensors are experimental devices constructed by directly or indirectly fixing reagents participating in a chemiluminescent reaction on a working electrode by a chemical modification method, and are called electrochemical luminescence sensors or electrochemiluminescence sensors. The electrochemiluminescence sensor not only maintains the advantage of electrochemiluminescence, but also overcomes the defects that the popularization and application of the electrochemiluminescence analysis method are limited due to poor selectivity and the use of a complex optical system and a solution-type electrochemiluminescence reagent in the conventional electrochemiluminescence analysis, widens the application range of the electrochemiluminescence analysis method, and realizes the miniaturization of the instrument and the increase of the practicability of the method. However, visual electrochemiluminescence is more suitable for in situ detection of mycotoxins than conventional ECL bioanalytical systems using photomultiplier tubes (PMTs), and no special instrumentation is required for imaging ECL signal visual sensing. 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, portability and the like, and has wide application in the field of environmental detection and food detection.
Ochratoxin a (OTA) is a secondary metabolite produced by a variety of aspergillus and penicillium fungi. The OTA has stable physicochemical properties, is widely applied to cereal related products such as corn, wheat, oat and the like, and is widely focused due to high toxicity. The ingestion of OTA toxins is a great threat to both humans and animals. Mainly damages hematopoietic organs, immune organs, digestive system, nervous system and reproductive system. In view of the harmfulness of OTA, one of the most dangerous naturally occurring food pollutants has been identified by the united nations grain and farming organizations and the world health organization. Currently, detection methods for OTA include enzyme-linked adsorption immunosorbent assay, electrochemical immunosensor detection, biosensor detection and the like. The detection method has the advantages of single detection mode, complex operation, portability and incapability of realizing on-site rapid detection. Therefore, it is necessary to develop an analysis method that is flexible, fast, simple and portable in detection mode and low in cost.
Disclosure of Invention
The invention aims to provide a portable visual electrochemiluminescence aptamer sensor which integrates the advantages of rapidness, simplicity, miniaturization, flexible use mode and the like, is applied to detection of OTA toxin, and utilizes BiOI to detect Ru (bpy) 3 2+ The fixed enhancement effect of (2) does not need to adopt a traditional Photomultiplier (PMT) amplification strategy, and two areas are designed on ITO, so that the visual unit and the detection unit are separated, and the electrochemical luminescence visual detection based on the change of the luminescence intensity of the ruthenium (II) complex luminophor is realized.
The construction method of the visual electrochemiluminescence sensing device comprises the following steps:
step 1, preparing a luminescent material bipyridine ruthenium-iodine bismuth oxide composite microsphere (Ru (bpy) 3 2+ /BiOI)：
Firstly, adding ruthenium bipyridine into bismuth oxyiodide dispersion liquid, and stirring to form a uniformly dispersed aqueous solution A; then, the aqueous solution A is transferred into an ultrasonic machine for ultrasonic treatment, so that Ru (bpy) is successfully prepared 3 2+ BiOI microsphere composite; finally, washing with water and ethanol for several times, and drying to prepare a solid product Ru (bpy) 3 2+ BiOI microsphere.
Step 2, preparing a visual electrode substrate
Sequentially ultrasonically cleaning ITO in toluene, acetone, ethanol and water, drying in nitrogen flow, then performing electrochemical cleaning on ITO glass by adopting a cyclic voltammetry to remove impurities and organic pollutants possibly adsorbed on the surface, and finally drying by using nitrogen; laser etching a visual area and a detection area on the clean ITO;
step 3, modifying the visualized area
The solid product Ru (bpy) obtained in step 1 was purified 3 2+ BiOI is dispersed in deionized water to obtain Ru (bpy) 3 2+ BiOI dispersion of Ru(bpy) 3 2+ The BiOI dispersion is dripped on the visualization area of ITO, nafion solution is dripped to form uniform film fixing material, and the film fixing material is placed in an oven for drying to obtain the visualization 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, preparing an OTA aptamer solution by using PBS as a solvent, dripping the OTA aptamer solution on a transparent film of graphene oxide, incubating for 10 hours at 4 ℃, leaching by using PBS to remove any unbound aptamer, and dripping a Bovine Serum Albumin (BSA) solution to block a nonspecific active site; finally obtaining aptamer/GO/ITO of the detection area modified by the aptamer;
step 5, constructing a visual electrochemiluminescence aptamer sensing device for detecting OTA toxin
aptamer/GO/ITO of detection area modified by aptamer obtained in step 4 and Ru (bpy) of visualization area in step 3 3 2+ The BiOI/Nafion forms a visual electrochemiluminescence aptamer sensing device.
In the step 1, the dosage ratio of the bipyridine ruthenium, the bismuth oxyiodide and the deionized water in the solution A is 0.375g:0.042g:50mL; the drying temperature is 60 ℃ and the drying time is 12 hours.
In the step 2, the ultrasonic time is 15min, the parameters of the cyclic voltammetry are set to be scanned 15 times in the range of 0-1.5V in 0.1mol/L sodium hydroxide solution, then the cyclic voltammetry is scanned 30 times in 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 10min.
In step 3, ru (bpy) 3 2+ The concentration of the BiOI dispersion is 2mg/mL, and the drop coating amount is 20 mu L; the mass percentage concentration of the Nafion solution is 0.5 percent, and the dripping amount is 10 mu L; the drying temperature is 60 ℃ and the drying time is 2h.
In the step 4, the mass percentage concentration of the Graphene Oxide (GO) solution is 0.2%, the dropwise adding amount is 20 mu L, and the standing time is 2h.
In the step 4 of the process, the process is carried out,
the concentration of the PBS solution is 0.1mol/L;
the OTA aptamer sequence is: 5'-GATC GGGT GTGG GTGG CGTA AAGG GAGC ATCG GACA A-3'; OTA aptamer concentration is 4 mu M, dropwise adding amount is 20 mu L, and incubation time is 10h; the mass percentage concentration of BSA was 3%, and the drop amount was 20. Mu.L.
The application of the visual electrochemiluminescence aptamer sensing device prepared by the invention in detecting ochratoxin OTA comprises the following specific steps:
(1) Dropping OTA solutions with different concentrations to a detection area on an aptamer/GO/ITO electrode, and incubating for a period of time at room temperature;
(2) Placing the electrode treated in the step (1) into PBS electrolyte containing butyl Diethanolamine (DBAE), connecting the electrode with an electrochemical workstation by using a conductive adhesive tape, and performing constant potential scanning by using Ru (bpy) at the electrochemical workstation 3 2+ the/BiOI luminous intensity is taken as an output signal; ru (bpy) 3 2+ The change of RGB value and OTA concentration are made into standard curve when BiOI emits light;
(3) And collecting signals from the OTA solution with unknown concentration by adopting the method, and substituting the signals into a 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 dropwise adding amount is 20 mu L;
in step (2), the concentration of DBAE is 20mM; the PBS content is 20-30 mL; the constant potential was 1.3V and the reaction time was 30s.
The beneficial effects of the invention are as follows:
the invention takes ITO with functional area as a substrate to modify Ru (bpy) 3 2+ The visual electrochemiluminescence aptamer sensor is successfully established by taking the area of the BiOI microsphere as a visual area and taking a modified Graphene Oxide (GO) area as a detection area, so that quantitative analysis and detection of OTA toxins are realized, and the characteristics and advantages are expressed as follows:
(1) Ru (bpy) is prepared according to the invention 3 2+ BiOI microspheres as visual area luminophores to modifyThe graphene oxide substrate is used as a detection area to construct a visual electrochemiluminescence aptamer sensor, and a luminescence signal is stable.
(2) Ru (bpy) is prepared according to the invention 3 2+ BiOI microsphere to enhance Ru (bpy) 3 2+ The electrode can realize visual detection of OTA toxin without using an electrochemiluminescence detection instrument, thereby reducing the complexity of detection and the cost.
(3) The visual electrochemiluminescence aptamer sensor provided by the invention realizes the sensitive detection of the OTA, and the concentration of the OTA and Ru (bpy) are within the concentration range of 1-100 ng/mL 3 2+ The change of RGB value when the BiOI emits light shows good linear relation, and the detection limit can reach 0.33ng/mL.
(4) The visualized electrochemiluminescence aptamer sensor constructed by the invention does not need to rely on the connection mode of a traditional photomultiplier tube (PMT) for detection. Based on the electrode provided by the invention, not only can outdoor portable luminescence detection be realized, but also high-sensitivity quantitative detection can be realized, the purpose of timely detection can be achieved, and the use mode is flexible.
Drawings
FIG. 1 is a mechanism diagram of a constructed visual electrochemiluminescence aptamer sensor;
FIG. 2 is a schematic view of the ITO electrode area of the present invention;
FIG. 3, ru (bpy) for preparation 3 2+ Scanning electron microscopy image (A) and X-ray diffraction image (B) of the BiOI microsphere; an X-ray photoelectron spectrum (C);
FIGS. 4 (A), (B) show the concentration of OTA and Ru (bpy) 3 2+ A graph of RGB value change when the BiOI microsphere emits light, and a graph of the result of the sensor selectivity test.
Detailed Description
The invention is described in detail below with reference to the drawings and examples of the specification, but the invention is not limited to these examples.
Fig. 1 is a mechanism diagram of detection of a constructed visualized electrochemiluminescence aptamer sensor, and fig. 2 is a schematic diagram of an ITO electrode area of the present 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 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 was gradually changed from the initially clear solution to an orange suspension. Transferring the stirred suspension into a 50mL polytetrafluoroethylene autoclave, reacting for 12 hours at the temperature of 140 ℃, washing a sample with ethanol and deionized water for 3 times respectively after the autoclave is naturally cooled to the room temperature, and finally drying for 12 hours in a 60 ℃ oven to obtain the BiOI microspheres.
Different amounts of Ru (bpy) 3 2+ Dispersing BiOI in water, ultrasonic treating for 2 hr, stirring and mixing for 6 hr, and oven drying at 60deg.C to obtain Ru (bpy) 3 2+ BiOI microsphere particles.
Characterization results As shown in FIG. 3, successful preparation of BiOI microspheroidal particles can be seen from SEM and XRD of panels A and B, and Ru (bpy) from panel C 3 2+ XPS of BiOI microsphere particle tests, the elemental composition of the BiOI microsphere particle is analyzed, and the preparation of the composite material is successfully proved.
(2) Electrode substrate pretreatment
The electrode of the invention is mainly completed in two parts, including the manufacture of the substrate layer and the manufacture of the reaction area. First, an ITO glass having a stripe pattern is formed by pattern etching. In short, firstly, acetone, ethanol and deionized water are sequentially used for cleaning the ITO surface, and nitrogen is dried for standby. And then, performing electrochemical cleaning on the ITO glass by adopting a cyclic voltammetry to remove impurities and organic pollutants possibly adsorbed on the surface, and using laser etching to display the pattern with the functional area on the clean ITO. Separate circular detection units (r=8mm) and visualization units (r=8mm) were formed on the ITO glass surface.
(3) Modified visual electrode
The working electrode area was fixed with r=8mm round brown high temperature resistant tape and 2mg Ru (bpy) was weighed out 3 2+ BiOI was dispersed in 1mL deionized water to give Ru (bpy) 3 2+ Transfer 20. Mu.L Ru/BiOI dispersionbpy) 3 2+ Uniformly dripping the BiOI dispersion liquid on the visual electrode area, continuously dripping 10 mu L of Nafion solution with the mass concentration of 0.5%, and drying in a 60 ℃ oven for 2 hours to obtain Ru (bpy) 3 2+ /BiOI/Nafion。
(4) Construction of visual electrochemiluminescence aptamer sensing device
And (3) spin-coating 20 mu L of Graphene Oxide (GO) solution on the electrode detection area, wherein the mass percentage concentration is 0.2%, and the standing time is 2h. A 4 μm solution of OTA aptamer was prepared using PBS (ph=7.4, 0.1 mol/L) as solvent, the OTA aptamer sequence being: 5'-GATC GGGT GTGG GTGG CGTA AAGG GAGC ATCG GACA A-3'. Dripping 20 mu L of OTA aptamer on an electrode detection area, placing in a refrigerator at 4 ℃ for reaction for 10 hours, leaching with PBS to remove excessive unadsorbed aptamer, dripping 20 mu L of 3% Bovine Serum Albumin (BSA) solution to block nonspecific active sites,
the resulting 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 sensing device for detecting OTA
20. Mu.L of OTA toxin at a concentration of 1,10,25,50,75 and 100ng/mL, respectively, was dropped onto the detection zone of the electrode and incubated at room temperature for 40min. Finally, the electrode was placed in an electrolytic cell with PBS (pH=7.4, 0.1 mol/L) at a concentration of 20mM butyl Diethanolamine (DBAE), and the amount of the solution was 30mL. The electrode and the electrochemical workstation are connected by using a conductive adhesive tape, the electrochemical workstation performs constant potential scanning, the potential is 1.3V, and the reaction time is 30s. Ru (bpy) 3 2+ the/BiOI luminous intensity is taken as an output signal; ru (bpy) 3 2+ The change in RGB value upon BiOI luminescence was analyzed chemically.
The detection results are shown in fig. 4, and the detection results of the sensor in the visualization mode under different OTA concentrations are shown in fig. 4. As can be seen from FIG. A, ru (bpy) increases with the concentration of OTA 3 2+ The more the color of the/BiOI emission, the greater the change in RGB values. As can be seen from FIG. B, in the concentration range of 1 to 100ng/mL,OTA concentration and Ru (bpy) 3 2+ The change of RGB value when the BiOI emits light shows good linear relation, the detection limit can reach 0.33ng/mL, and the sensor has good selectivity as shown in a graph C.
Claims (9)
1. The construction method of the visualized electrochemical luminescence sensor based on the ruthenium (II) complex is characterized by comprising the following steps:
step 1, preparing a luminescent material bipyridine ruthenium-iodine bismuth oxide composite microsphere Ru (bpy) 3 2+ /BiOI：
Firstly, adding ruthenium bipyridine into bismuth oxyiodide dispersion liquid, and stirring to form a uniformly dispersed aqueous solution A; then, the aqueous solution A is transferred into an ultrasonic machine for ultrasonic treatment, so that Ru (bpy) is successfully prepared 3 2+ BiOI microsphere composite; finally, washing and drying are carried out, thus preparing a solid product Ru (bpy) 3 2+ BiOI microspheres;
step 2, preparing a visual electrode substrate
Sequentially ultrasonically cleaning ITO in toluene, acetone, ethanol and water, drying in nitrogen flow, then performing electrochemical cleaning on ITO glass by adopting a cyclic voltammetry to remove impurities and organic pollutants possibly adsorbed on the surface, and finally drying by using nitrogen; laser etching a visual area and a detection area on the clean ITO;
step 3, modifying the visualized area
The solid product Ru (bpy) obtained in step 1 was purified 3 2+ BiOI is dispersed in deionized water to obtain Ru (bpy) 3 2+ BiOI dispersion, ru (bpy) 3 2+ The BiOI dispersion is dripped on the visualization area of ITO, nafion solution is dripped to form uniform film fixing material, and the film fixing material is placed in an oven for drying to obtain the visualization electrode Ru (bpy) 3 2+ a/BiOI/Nafion electrode;
step 4, modifying the detection area:
firstly, spin-coating graphene oxide GO solution in a detection area, and standing at room temperature to form a transparent film;
then, using PBS as a solvent to prepare an OTA aptamer solution, dripping the OTA aptamer solution on a transparent film of graphene oxide, incubating for a certain time, leaching with PBS to remove any unbound aptamer, and dripping a bovine serum albumin BSA solution to block non-specific active sites; finally obtaining aptamer/GO/ITO of the detection area modified by the aptamer;
OTA is Hexarotoxin A;
step 5, constructing a visual electrochemiluminescence aptamer sensing device for detecting OTA toxin
aptamer/GO/ITO of detection area modified by aptamer obtained in step 4 and Ru (bpy) of visualization area in step 3 3 2+ The BiOI/Nafion forms a visual electrochemiluminescence aptamer sensing device.
2. The method of claim 1, wherein in step 1, the solution a contains ruthenium bipyridyl, bismuth oxyiodide and deionized water in an amount ratio of 0.375g:0.042g:50mL; the drying temperature is 60 ℃ and the drying time is 12 hours.
3. The method of claim 1, wherein in step 2, the ultrasonic wave is performed for 15 minutes, the cyclic voltammetry is set to be performed 15 times in the range of 0-1.5V in 0.1mol/L sodium hydroxide solution, then the cyclic voltammetry is performed 30 times in the range of 0-1.6V in 0.5mol/L sulfuric acid solution, the scanning speed is 100mV/s, the time for introducing high-purity nitrogen gas is 10 minutes, and the concentration of the high-purity nitrogen gas is 99.999%.
4. The method of claim 1, wherein in step 3, ru (bpy) 3 2+ The concentration of the BiOI dispersion is 2mg/mL, and the drop coating amount is 20 mu L; the mass percentage concentration of the Nafion solution is 0.5 percent, and the dripping amount is 10 mu L; the drying temperature is 60 ℃ and the drying time is 2h.
5. The construction method according to claim 1, wherein in step 4, the mass percentage concentration of the Graphene Oxide (GO) solution is 0.2%, the drop amount is 20 μl, and the standing time is 2h.
6. The construction method according to claim 1, wherein,
in the step 4, the concentration of the PBS solution is 0.1mol/L;
the OTA aptamer sequence is: 5'-GATC GGGT GTGG GTGG CGTA AAGG GAGC ATCG GACA A-3'; OTA aptamer concentration is 4 μM, and the drop amount is 20 μL; incubation temperature is 4 ℃ and time is 10 hours; the mass percentage concentration of BSA was 3%, and the drop amount was 20. Mu.L.
7. Use of a visualized electrochemiluminescence aptamer sensor device constructed according to the construction method of any one of claims 1-6 for detecting ochratoxin a.
8. The method according to claim 7, wherein the specific step of detecting is
(1) Dropping OTA solutions with different concentrations to a detection area on an aptamer/GO/ITO electrode, and incubating for a period of time at room temperature;
(2) Placing the electrode treated in the step (1) into PBS electrolyte containing butyl diethanolamine DBAE, connecting the electrode with an electrochemical workstation by using a conductive adhesive tape, and performing constant potential scanning by using Ru (bpy) at the electrochemical workstation 3 2+ the/BiOI luminous intensity is taken as an output signal; ru (bpy) 3 2+ The change of RGB value and OTA concentration are made into standard curve when BiOI emits light;
(3) And collecting signals from the OTA solution with unknown concentration by adopting the method, and substituting the signals into a standard curve to obtain the concentration of the OTA solution.
9. The use according to claim 8, wherein,
in the step (1), the concentration of OTA is 1-100 ng/mL, and the dropwise adding amount is 20 mu L;
in step (2), the concentration of DBAE is 20mM; the PBS content is 20-30 mL; the constant potential was 1.3V and the reaction time was 30s.
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