A preparation method of a photo-polymerizable ligand photoelectrochemical sensor

Abstract

The present invention relates to the field of biochemical electrode technology, and specifically to a method for preparing a photopolymerized aptamer photoelectrochemical sensor. The method of combining an aptamer with a semiconductor photosensitive material and modifying it on the surface of a photoelectrode by conventional electrostatic adsorption or chemical crosslinking is inefficient. In view of the above problems, the present invention provides a method for preparing a photopolymerized aptamer photoelectrochemical sensor, which uses photopolymerization technology to modify noble metal nanoparticles and aptamers on the surface of a photoelectrode to obtain a photoelectrochemical sensor. The method is rapid, efficient, and has good reproducibility. The surface of the obtained photoelectrochemical sensor is composed of a semiconductor photosensitive material layer, a noble metal nanoparticle layer, and an aptamer binding layer. This layered structure makes the distance between the noble metal nanoparticles and the semiconductor photosensitive material closer, and can better exert the effect of noble metal nanoparticle plasma signal enhancement.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical electrodes, and particularly relates to a preparation method of a photopolymerizable ligand photoelectrochemical sensor. Background Art

[0002] A photoelectrochemical biosensor is a sensor that combines a biological recognition element based on photoelectrochemistry. Commonly used biological recognition elements include enzymes, antigen-antibody, aptamers, etc. Among them, an aptamer is a single-stranded oligonucleotide that can specifically bind to ions, small molecules, proteins or other target substances obtained by in vitro screening technology of systematic evolution of ligands by exponential enrichment. It has various three-dimensional structures. When the target substance exists, its own conformation will change, and through shape complementarity, electrostatic interaction or π-bond stacking, etc., an aptamer-target substance complex is formed, showing the advantages of high selectivity and strong interaction. In addition, aptamers also have many advantages such as stable chemical properties, can be artificially screened and synthesized in vitro by chemical methods, and are easy to modify in structure. Their applications have extended to many fields such as video security, medical diagnosis, drug carriers, materials science, environmental monitoring, etc.

[0003] The common method for constructing a photoelectrochemical aptamer sensor is first to modify a photosensitive material on the electrode surface, and then bind the aptamer to the semiconductor photosensitive material by electrostatic adsorption or chemical cross-linking. Although the above methods are relatively mature, the binding efficiency needs to be further improved.

[0004] The photopolymerization technology is a method for preparing polymer materials by photoinitiated polymerization. It has a fast reaction rate, mild conditions, less energy required, and unique time and space controllability. The present invention utilizes the fast, mild and easy-to-control characteristics of the photopolymerization reaction, combined with the photoreduction characteristics, to construct a photopolymerizable ligand encapsulated photosensitive material and a photoelectrochemical aptamer sensor with in-situ enhanced electrical signals by noble metal nanoparticles. During the construction process, by controlling the content of the aptamer, the concentration of the initiator and the concentration of noble metal ions, the grafting density of the aptamer and the reduced particle size of the noble metal nanoparticles can be regulated. This new method for constructing a photoelectrochemical aptamer sensor has important potential application value for realizing the rapid, controllable and batch preparation of photoelectrochemical aptamers. Summary of the Invention

[0005] The problem existing in the prior art is that the method of binding an aptamer to a semiconductor photosensitive material by conventional electrostatic adsorption or chemical cross-linking and modifying it on the surface of a photoelectrode has low efficiency. To solve the above problems, the present invention provides a preparation method of a photopolymerizable ligand photoelectrochemical sensor, including the following preparation steps:

[0006] (1) Coat a solution containing a semiconductor photosensitive material on the surface of an ITO optoelectrode, and then dry the electrode surface to obtain a semiconductor photosensitive material modified electrode;

[0007] (2) Coat a photopolymerization solution containing a noble metal ion on the surface of the semiconductor photosensitive material modified electrode obtained in step (1), and then perform UV irradiation on the electrode surface to obtain a noble metal nanoparticle modified optoelectrode;

[0008] (3) Coat a photopolymerization solution containing an aptamer on the surface of the noble metal nanoparticle modified optoelectrode obtained in step (2), and then perform UV irradiation on the electrode surface to obtain a photopolymerized aptamer optoelectrochemical sensor.

[0009] Specifically, the semiconductor photosensitive material includes at least one of titanium dioxide, zinc oxide, bismuth sulfide, and cadmium sulfide.

[0010] Specifically, the solution of the semiconductor photosensitive material in step (1) is composed of a semiconductor photosensitive material and a solvent, and the mass-volume ratio of the semiconductor photosensitive material to the solvent is 1.0 mg: 1 mL.

[0011] Specifically, the coating density of the semiconductor photosensitive material on the electrode surface is 0.07 - 0.14 mg / cm 2 .

[0012] Specifically, the photopolymerization solution containing a noble metal ion in step (2) includes the following components by weight:

[0013]

[0014] Specifically, the noble metal salt includes at least one of silver nitrate, chloroauric acid, chloroplatinic acid, and palladium chloride.

[0015] Specifically, the photopolymerization solution containing an aptamer includes the following components by weight:

[0016]

[0017] Specifically, the aptamer includes at least one of acrylamide-modified heavy metal ion aptamer, acrylamide-modified mycotoxin aptamer, and acrylamide-modified antibiotic aptamer.

[0018] Specifically, the cross-linking agent includes at least one of bisacrylamide and polyethylene glycol diacrylate.

[0019] Specifically, the photoinitiator includes at least one of photoinitiator 2959, photoinitiator 1173, photoinitiator 184, and photoinitiator TPO.

[0020] Specifically, the solvent includes water or absolute ethanol.

[0021] Beneficial effects

[0022] (1) The method for obtaining an aptamer photoelectrochemical sensor by using the rapid photopolymerization technology in the present invention is fast, efficient, and has good reproducibility.

[0023] (2) The surface of the photoelectrochemical sensor obtained in the present invention is composed of a semiconductor photosensitive material layer, a noble metal nanoparticle layer, and an aptamer binding layer. This layered structure makes the distance between the noble metal nanoparticles and the semiconductor photosensitive material closer, and can better exert the effect of enhancing the plasmon signal of the noble metal nanoparticles.

[0024] (3) During the photopolymerization process of the photopolymerization solution containing noble metal ions, its surface will be affected by oxygen inhibition of polymerization, resulting in the partial crosslinking agent on the surface of the photopolymerization solution not being able to crosslink and cure, and serving as the active binding points of the aptamer layer. When the photopolymerization solution containing aptamer is coated on the surface of the noble metal nanoparticle layer and placed under light irradiation, the crosslinking agent of the noble metal nanoparticle layer will participate in the photocuring process of the photopolymerization solution containing aptamer, thereby firmly fixing the aptamer on the electrode surface. In addition, when the photopolymerization solution containing aptamer is completely cured, the noble metal nanoparticle layer is completely wrapped between the aptamer layer and the semiconductor photosensitive material layer, and the noble metal nanoparticles are not easily detached, and the properties of the obtained photoelectrochemical sensor are more stable. Description of the drawings

[0025] Figure 1 : It is a graph of the photocurrent response signal - time curve of the modified electrode obtained in Example 1. Detailed implementation manners

[0026] Example 1

[0027] A preparation method of a photopolymerized ligand photoelectrochemical sensor, the preparation steps are as follows:

[0028] (1) Coat a semiconductor photosensitive material solution with a mass concentration of 1 mg / mL on the surface of an ITO photoelectrode with a diameter of 0.6 cm, and the coating amount is 20 μL. Then dry the electrode surface to obtain a semiconductor photosensitive material modified electrode.

[0029] (2) Coat a photopolymerization solution containing noble metal ions on the surface of the semiconductor photosensitive material modified electrode obtained in step (1), and the coating amount is 20 μL. Then irradiate the electrode surface with UV light to obtain a noble metal nanoparticle modified photoelectrode. The photopolymerization solution containing noble metal ions is composed of the following components by weight:

[0030] Silver nitrate 20%

[0031] Bisacrylamide 5%

[0032] Photoinitiator 2959 5%

[0033] Absolute ethanol 70%;

[0034] (3) Coating the surface of the noble metal nanoparticle modified optoelectrode obtained in step (2) with a photopolymerization solution containing aptamer, the coating amount is 20 μL, and then irradiating the electrode surface with UV light to obtain a photopolymerized aptamer optoelectrochemical sensor. The photopolymerization solution containing aptamer is composed of the following components by weight:

[0035] Aptamer 20%

[0036] Bisacrylamide 5%

[0037] Photoinitiator 2959 5%

[0038] Absolute ethanol 70%.

[0039] The aptamer is purchased from Sangon Biotech (Shanghai) Co., Ltd., and its (for identifying Pb 2+ ) has the following structural formula:

[0040] CH 2 =CH-CO-NH-5'-(CH 2 ) 6 -GGT TGG TGT GGT TGG-3'.

[0041] Example 2

[0042] A preparation method of a photopolymerized aptamer optoelectrochemical sensor, the preparation steps are as follows:

[0043] (1) Coating a semiconductor photosensitive material solution with a mass concentration of 1 mg / mL on the surface of an ITO optoelectrode with a diameter of 0.6 cm, the coating amount is 20 μL, and then drying the electrode surface to obtain a semiconductor photosensitive material modified electrode;

[0044] (2) Coating the surface of the semiconductor photosensitive material modified electrode obtained in step (1) with a photopolymerization solution containing noble metal ions, the coating amount is 20 μL, and then irradiating the electrode surface with UV light to obtain a noble metal nanoparticle modified optoelectrode. The photopolymerization solution containing noble metal ions is composed of the following components by weight:

[0045] Chloroauric acid 20%

[0046] Polyethylene glycol diacrylate 5%

[0047] Photoinitiator 1173 5%

[0048] Absolute ethanol 70%;

[0049] (3) Coat the surface of the noble metal nanoparticle-modified optoelectrode obtained in step (2) with a photopolymerizable solution containing an aptamer, with a coating amount of 20 μL, and then perform UV light irradiation on the electrode surface to obtain a photopolymerizable aptamer photoelectrochemical sensor. The photopolymerizable solution containing an aptamer is composed of the following components by weight:

[0050] Aptamer 20%

[0051] Polyethylene glycol diacrylate 5%

[0052] Photoinitiator 1173 5%

[0053] Absolute ethanol 70%.

[0054] The aptamer is purchased from Sangon Biotech (Shanghai) Co., Ltd., and its structural formula (for identifying aflatoxin AFB1) is as follows:

[0055] CH 2 =CH-CO-NH-5’-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCG CTAGGCCCACA-3’.

[0056] Example 3

[0057] A preparation method of a photopolymerizable aptamer photoelectrochemical sensor, and the preparation steps are as follows:

[0058] (1) Coat a semiconductor photosensitive material solution with a mass concentration of 1 mg / mL on the surface of an ITO optoelectrode with a diameter of 0.6 cm, with a coating amount of 20 μL, and then dry the electrode surface to obtain a semiconductor photosensitive material-modified electrode;

[0059] (2) Coat the surface of the semiconductor photosensitive material-modified electrode obtained in step (1) with a photopolymerizable solution containing noble metal ions, with a coating amount of 20 μL, and then perform UV light irradiation on the electrode surface to obtain a noble metal nanoparticle-modified optoelectrode. The photopolymerizable solution containing noble metal ions is composed of the following components by weight:

[0060] Silver nitrate 20%

[0061] Polyethylene glycol diacrylate 5%

[0062] Photoinitiator 1173 5%

[0063] Absolute ethanol 70%;

[0064] (3) Coating a photopolymerization solution containing aptamer on the surface of the noble metal nanoparticle-modified optoelectrode, with a coating amount of 20 μL, and then performing UV light irradiation on the electrode surface to obtain a photopolymerized aptamer-based photoelectrochemical sensor. The photopolymerization solution containing aptamer has the following composition by weight:

[0065] Aptamer 20%

[0066] Polyethylene glycol diacrylate 5%

[0067] Photoinitiator 1173 5%

[0068] Water 70%.

[0069] The aptamer is purchased from Sangon Biotech (Shanghai) Co., Ltd., and its structural formula (for identifying oxytetracycline OTC) is as follows:

[0070] CH 2 =CH-CO-NH-5’-CGTACGGAATTCGCTAGCGGGCGGGGGTGCTGGGGGAATGGAGTGCTGCGTGCTGCGGGGATCCGAGCTCCACGTG-3’.

[0071] Example 4

[0072] A preparation method of a photopolymerized aptamer-based photoelectrochemical sensor, and the preparation steps are as follows:

[0073] (1) Coating a semiconductor photosensitive material solution with a mass concentration of 1 mg / mL on the surface of an ITO optoelectrode with a diameter of 0.6 cm, with a coating amount of 30 μL, and then drying the electrode surface to obtain a semiconductor photosensitive material-modified electrode;

[0074] (2) Coating a photopolymerization solution containing noble metal ions on the surface of the semiconductor photosensitive material-modified electrode obtained in step (1), with a coating amount of 30 μL, and then performing UV light irradiation on the electrode surface to obtain a noble metal nanoparticle-modified optoelectrode. The photopolymerization solution containing noble metal ions has the following composition by weight:

[0075] Chloroauric acid 20%

[0076] Bisacrylamide 5%

[0077] Photoinitiator TPO 5%

[0078] Absolute ethanol 70%;

[0079] (3) Coat the surface of the optoelectrode modified with noble metal nanoparticles with a photopolymerization solution containing aptamer. The coating amount is 30 μL. Then, irradiate the electrode surface with UV light to obtain a photopolymerized aptamer photoelectrochemical sensor. The photopolymerization solution containing aptamer is composed of the following components by weight:

[0080] Aptamer 20%

[0081] Bisacrylamide 5%

[0082] Photoinitiator TPO 5%

[0083] Absolute ethanol 70%.

[0084] The aptamer is purchased from Sangon Biotech (Shanghai) Co., Ltd. The structural formula for (identifying aflatoxin AFB1) is as follows:

[0085] CH 2 =CH-CO-NH-5’-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’.

[0086] Example 5

[0087] A preparation method of a photopolymerized aptamer photoelectrochemical sensor, the preparation steps are as follows:

[0088] (1) Coat the surface of an ITO optoelectrode with a diameter of 0.6 cm with a semiconductor photosensitive material solution with a mass concentration of 1 mg / mL. The coating amount is 40 μL. Then, dry the electrode surface to obtain a semiconductor photosensitive material modified electrode;

[0089] (2) Coat the surface of the semiconductor photosensitive material modified electrode obtained in step (1) with a photopolymerization solution containing noble metal ions. The coating amount is 40 μL. Then, irradiate the electrode surface with UV light to obtain a noble metal nanoparticle modified optoelectrode. The photopolymerization solution containing noble metal ions is composed of the following components by weight:

[0090] Chloroauric acid 20%

[0091] Bisacrylamide 5%

[0092] Photoinitiator 184 5%

[0093] Absolute ethanol 70%;

[0094] (3) Coat the surface of the optoelectrode modified with noble metal nanoparticles with the aptamer-containing photopolymerization solution, and the coating amount is 40 μL. Then, irradiate the electrode surface with UV light to obtain the photopolymerized aptamer photoelectrochemical sensor. The aptamer-containing photopolymerization solution is composed of the following components by weight:

[0095] Aptamer 20%

[0096] Bisacrylamide 5%

[0097] Photoinitiator 184 5%

[0098] Absolute ethanol 70%.

[0099] The aptamer is purchased from Sangon Biotech (Shanghai) Co., Ltd., and its structural formula (for identifying aflatoxin AFB1) is as follows:

[0100] CH 2 =CH-CO-NH-5’-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’.

[0101] Comparative Example 1 is the same as Example 1, except that in Comparative Example 1, 20 μL of the photopolymerization solution containing noble metal ions obtained in step (2) of Example 1 and 20 μL of the aptamer-containing photopolymerization solution obtained in step (3) of Example 1 are taken, mixed evenly, and then all are coated on the surface of the semiconductor photosensitive material modified electrode obtained in step (1). After that, the electrode surface is irradiated with UV light to obtain the aptamer-modified electrode.

[0102] Comparative Example 2 is the same as Example 1, except that in Comparative Example 2, 20 μL of the semiconductor photosensitive material solution in step (1) of Example 1 and 20 μL of the photopolymerization solution containing noble metal ions obtained in step (2) of Example 1 are taken, mixed evenly, and then all are coated on the surface of the ITO optoelectrode. After that, the electrode surface is irradiated with UV light to obtain the noble metal nanoparticle-modified optoelectrode.

[0103] Performance test

[0104] Perform relevant performance tests on the modified electrodes obtained in Examples 1-5 and Comparative Examples 1-2 of the present invention. The test method is as follows:

[0105] A three - electrode system (a platinum wire (Pt) as the counter electrode, an Ag / AgCl (saturated KCl solution) electrode as the reference electrode, and ITO as the working electrode) was adopted. The photocurrent - time (i - t) curve test was carried out using a photoelectrochemical (PEC) test device composed of a CHI - 660E electrochemical workstation and a xenon light source (PLS - SXM300 / 300UV). The time for switching the light was set at intervals of 20 s. According to the amplitude of the change in the photocurrent signal before and after adding a certain concentration of the target substance, its detection sensitivity was determined. A large signal change indicates high sensitivity. Among them, the background photocurrent response signal diagram of the modified electrode obtained in Example 1 is shown in the appendix Figure 1 as follows

[0106] Among them, the specific test results are shown in Table 1

[0107] Table 1

[0108] Test item Photocurrent response Example 1 1500 nA Example 2 1400 nA Example 3 1100 nA Example 4 1050 nA Example 5 960 nA Comparative example 1 800 nA Comparative example 2 750 nA

[0109] As mentioned above, it is only a preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art, within the scope of the technical solution of the present invention, can make some changes or modifications to equivalent embodiments by using the above - disclosed technical content. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention

Claims

1. A preparation method of a photo-polymerizable aptamer photoelectrochemical sensor, characterized in that, it includes the following preparation steps: (1) Coat a solution containing a semiconductor photosensitive material on the surface of an ITO photoelectrode, and then dry the electrode surface to obtain a semiconductor photosensitive material modified electrode; (2) Coat a photo-polymerization solution containing noble metal ions on the surface of the semiconductor photosensitive material modified electrode obtained in step (1), and then perform UV light irradiation on the electrode surface to obtain a noble metal nanoparticle modified photoelectrode; (3) Coat a photo-polymerization solution containing an aptamer on the surface of the noble metal nanoparticle modified photoelectrode obtained in step (2), and then perform UV light irradiation on the electrode surface to obtain a photo-polymerizable aptamer photoelectrochemical sensor; The photo-polymerization solution containing noble metal ions described in step (2) includes the following components by weight: Noble metal salt 20% Cross-linking agent 5% Photoinitiator 5% Solvent 70%; The photo-polymerization solution containing an aptamer includes the following components by weight: Aptamer 20% Cross-linking agent 5% Photoinitiator 5% Solvent 70%; The cross-linking agent includes at least one of bisacrylamide and polyethylene glycol diacrylate.

2. The preparation method of a photo-polymerizable aptamer photoelectrochemical sensor according to claim 1, characterized in that, the semiconductor photosensitive material includes at least one of titanium dioxide, zinc oxide, bismuth sulfide, and cadmium sulfide.

3. The preparation method of a photo-polymerizable aptamer photoelectrochemical sensor according to claim 1, characterized in that, the solution of the semiconductor photosensitive material described in step (1) is composed of a semiconductor photosensitive material and a solvent, and the mass-volume ratio of the semiconductor photosensitive material to the solvent is 1.0 mg: 1 mL.

4. The preparation method of a photo-polymerizable aptamer photoelectrochemical sensor according to claim 1, characterized in that, The coating density of the semiconductor photosensitive material on the electrode surface is 0.07 - 0.14 mg / cm 2 .

5. The preparation method of a photo-polymerizable aptamer photoelectrochemical sensor according to claim 1, characterized in that, the noble metal salt includes at least one of silver nitrate, chloroauric acid, chloroplatinic acid, and palladium chloride.

6. The preparation method of a photo-polymerizable aptamer photoelectrochemical sensor according to claim 1, characterized in that, the aptamer includes at least one of acrylamide-modified heavy metal ion aptamer, acrylamide-modified mycotoxin aptamer, and acrylamide-modified antibiotic aptamer.

7. The preparation method of a photo-polymerizable aptamer photoelectrochemical sensor according to claim 1, characterized in that, the photoinitiator includes at least one of photoinitiator 2959, photoinitiator 1173, photoinitiator 184, and photoinitiator TPO.

8. The preparation method of a photo-polymerizable aptamer photoelectrochemical sensor according to claim 1 or 3, characterized in that, the solvent includes water or absolute ethanol.

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