CN112858412B - By using Bi 2 Sn 2 O 7 /Bi 2 S 3 Heterojunction-sensitized photoinduced electrochemical biosensor and application thereof - Google Patents
By using Bi 2 Sn 2 O 7 /Bi 2 S 3 Heterojunction-sensitized photoinduced electrochemical biosensor and application thereof Download PDFInfo
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Abstract
The invention discloses a Bi-based material 2 Sn 2 O 7 /Bi 2 S 3 A heterojunction sensitized photoelectrochemical biosensor and application thereof; the technical scheme of the invention is that DNA hydrogel with target response is utilized to coat sulfur ions, a large number of product chains are released by combining with exonuclease-assisted cyclic amplification reaction, the product chains are used for opening the DNA hydrogel to quantitatively release the sulfur ions, and the product chains are combined with a substrate material Bi 2 Sn 2 O 7 Anion exchange reaction to form Bi 2 Sn 2 O 7 /Bi 2 S 3 And heterojunction, and a photoelectrochemical biosensor is constructed to realize the analysis and detection of the target tumor cancer suppressor gene P53. The research has great application potential in the fields of biochemical analysis and clinical detection.
Description
The technical field is as follows:
the invention relates to a Bi 2 Sn 2 O 7 /Bi 2 S 3 A heterojunction-sensitized photoelectrochemical biosensor; and a preparation method of the photoelectrochemistry biosensor and an analysis application of the photoelectrochemistry biosensor in detecting tumor suppressor gene P53.
Background art:
photoelectrochemical (PEC) biosensors are a new detection technology developed on the basis of photoelectrochemistry, and have been widely used for sensitive detection of various biomarkers. The performance of the biosensor depends on the use of a semiconductor photosensitive material. The single semiconductor photon-generated carrier has high recombination speed and low absorption efficiency to visible light, and limits the practical application of the single semiconductor photon-generated carrier, so that a heterojunction is constructed to improve the performance of the sensor [ Kang, z; yan, x.; wang, y.; zhao, y.; bai, z; liu, y.; zhao, k.; cao, s.; zhang, y.nano.res.2015,9,344-352 ]. Hydrogel has advantages of ideal porous structure, expansion performance, mechanical strength and the like, so that the hydrogel is widely concerned as a potential sensing material for biosensor preparation [ Huang, Y.S.; ma, y.l.; chen, y.h.; wu, x.m.; fang, l.t.; zhu, z.; yang, C.J.anal.chem.2014,86,11434-11439. In recent years, nucleic acid analysis has been playing an increasingly important role in the fields related to tumor diagnosis, food hygiene, and the like. Especially P53, is one of the most important tumor suppressor genes due to its dysfunction in most human cancers [ Rasquinha, j.a.; bej, a.; dutta, s.; mukherjee, S.biochemistry,2017,56,4962-4971 ], and therefore, the selection of P53 as a detection target is of great significance.
DNA hydrogel is adopted to coat sulfur ions, a large number of product chains are obtained through exonuclease three-assisted target cyclic amplification, the product chains are used for opening the hydrogel to release the sulfur ions, and then the product chains and a substrate material bismuth stannate undergo an anion exchange reaction to obtain Bi 2 Sn 2 O 7 /Bi 2 S 3 The heterojunction enhances photoelectric signals, and a sensitive, simple, convenient and practical photoelectrochemical biosensor is prepared and used for detecting tumor cancer suppressor genes P53.
The invention content is as follows:
one of the objects of the present invention is to use a DNA hydrogel for the first time in a photoelectrochemical biosensor.
The preparation of the gel specifically comprises the following steps:
preparation of DNA hydrogel. mu.L of 100. Mu.M acrylamide-based modified DNA strand A and DNA strand B was added to 94. Mu.L of the stock solution. The stock solution contained 10mM Tris,1mM EDTA,140mM NaCl,5mM KCl,1mM CaCl 2 1mM MgCl 2 And 3% by mass of acrylamide. With N 2 The solution is deoxidized for 15min, and then 1.5 mu L of freshly prepared initiator ammonium persulfate and 3 mu L of accelerator TEMED are added for reaction for 5min to generate polyacrylamide chains P-A and P-B. Then, 2. Mu.L of 100. Mu.M ligated DNA and sodium sulfide at a final concentration of 50mM were added to form a DNA hydrogel.
The second purpose of the invention is to utilize carrier silicon dioxide microspheres and exonuclease to assist target circulation amplification to obtain a large number of product chains as a substitute target.
The method specifically comprises the following steps:
Step 2.Exo III auxiliary target circulation amplification process: mu.L of 1. Mu.M PD DNA and 10. Mu.L of SiO 2 incubating-CP at 37 deg.C for 2h to obtain SiO 2 And (4) adding targets with different concentrations and 10U of exonuclease III into the solution, incubating for 2 hours at 37 ℃ to obtain a large amount of product chain PD, then incubating for 30min at 70 ℃ to inactivate the exonuclease III, centrifuging, and taking the supernatant at 4 ℃ for later use.
The third purpose of the invention is to construct Bi by using anion exchange reaction for the first time 2 Sn 2 O 7 /Bi 2 S 3 A heterojunction photoelectrochemical biosensor realizes the analysis and application of the sensor to the detection of tumor suppressor gene P53.
The method specifically comprises the following steps:
step 1.Bi 2 Sn 2 O 7 The preparation of (1): 1.0g of SnCl 4 ·5H 2 O was dissolved in 22mL water and stirred for 15min, then the pH of the solution was adjusted to 6 with sodium hydroxide, at which time a large amount of white suspension was produced. After centrifugation, the white precipitate was mixed with 2.2g Bi (NO) 3 ) 3 ·5H 2 O, mixing, redispersing into 45mL of secondary water, adjusting the pH of the solution to 12 by using sodium hydroxide, transferring the solution into a high-pressure reaction kettle, reacting for 24 hours at 180 ℃, then centrifugally purifying by using deionized water for multiple times, and drying at 80 ℃ for later use.
Description of the drawings:
FIG. 1 DNA hydrogel based on target response with Bi 2 Sn 2 O 7 /Bi 2 S 3 A schematic of a heterojunction PEC biosensor for DNA detection.
FIG. 2Bi 2 Sn 2 O 7 The characterization of (1): XRD spectrum, (B) infrared spectrum, (C) ultraviolet spectrum, and (D) photoelectric signal pattern
FIG. 3 (A) Bi 2 Sn 2 O 7 /Bi 2 S heterojunction and Bi 2 Sn 2 O 7 、Bi 2 S 3 XPS spectrum comparison of (B) S2S spectrum, (C) Bi 2 Sn 2 O 7 EDS analysis of (D) heterojunction Bi 2 Sn 2 O 7 /Bi 2 S 3 EDS analysis of (3).
FIGS. 4 (A) and (B) show Bi before ion exchange reaction 2 Sn 2 O 7 SEM picture of (1); (C) (D) is Bi formed after the ion exchange reaction 2 Sn 2 O 7 /Bi 2 S 3 SEM image of heterojunction.
FIG. 5 (A) Bi 2 Sn 2 O 7 Schematic diagram of the transfer of photogenerated carriers; (B) Heterojunction Bi 2 Sn 2 O 7 /Bi 2 S 3 Schematic diagram of the transfer of photogenerated carriers.
FIG. 6 electrophoretic characterization: (A) an exonuclease III assists in a target cyclic amplification process; (B) a DNA gel forming process.
FIG. 7 (A) shows the photocurrent signal response curves at different target concentrations, 0, 10fM, 100fM, 1.0pM, 10pM, 100pM, 1.0nM, 10nM (from a to h), and the inset shows the comparison of the photocurrent signals at the target concentrations of 0 and 10 nM; (B) The linear relationship between the Δ I photocurrent variation and the concentration of the target.
The specific implementation mode is as follows:
example 1 Using DNA hydrogel and Bi 2 Sn 2 O 7 /Bi 2 S 3 A photoelectrochemical biosensor is developed by a heterojunction, and sensitive detection of a tumor suppressor gene P53 is realized.
mu.L of 1. Mu.M PD DNA and 10. Mu.L of SiO 2 -CP incubation at 37 ℃ for two hours to obtain SiO 2 And (4) adding targets with different concentrations and 10U of exonuclease III into the solution, incubating at 37 ℃ for 2h to obtain a large number of product chains PD, then incubating at 70 ℃ for 30min to inactivate the exonuclease III, centrifuging, and taking the supernatant at 4 ℃ for later use.
mu.L of 100. Mu.M acrylamide-based modified DNA strand A and DNA strand B was added to 94. Mu.L of the stock solution. The stock solution contained 10mM Tris,1mM EDTA,140mM NaCl,5mM KCl,1mM CaCl 2 1mM MgCl 2 And 3% by mass of acrylamide. With N 2 The solution is deoxidized for 15min, then 1.5 mul of freshly prepared initiator ammonium persulfate and 3 mul of accelerator TEMED are added, and the reaction is carried out for 5min to generate polyacrylamide chains P-A and P-B. Then, 2. Mu.L of 100. Mu.M ligated DNA and sodium sulfide at a final concentration of 50mM were added to form a DNA hydrogel.
Ultrasonically dispersing 0.5mg of bismuth stannate powder into 1mL of secondary water containing 0.5% of chitosan, and dripping 10 mu L of bismuth stannate solution onto an ITO electrode (control area: 0.16 cm) 2 ) And dried at room temperature as a base material. The resulting product chains PD at different target concentrations were then added to 10 μ l dna hydrogel and incubated for 1h for gel opening. Then 10 mu L of gel supernatant is dripped on the surface of the bismuth stannate material on the electrode, and the bismuth stannate material is kept wet for 10min at 37 ℃ to complete anion exchange reaction to form Bi 2 Sn 2 O 7 /Bi 2 S 3 And (3) rinsing the heterojunction with secondary water to remove unreacted sulfur ions, and naturally drying the heterojunction for PEC detection.
Example 2 Using DNA hydrogel and Bi 2 Sn 2 O 7 /Bi 2 S 3 A photoelectrochemical biosensor is developed by a heterojunction, and sensitive detection of a tumor suppressor gene P53 is realized.
"different concentrations of target and 10U of exonuclease III were added to the above solution and incubated at 37 ℃ for 2h. The other conditions of preparation of the target with different concentrations and 15U of exonuclease III are added into the solution and incubated for 2h at 37 ℃ are the same as example 1, and the biosensor with the appearance and the property similar to example 1 is obtained. The results of P53 detection were the same as in example 1.
Example 3 use of DNA hydrogel and Bi 2 Sn 2 O 7 /Bi 2 S 3 A photoelectrochemical biosensor is developed by a heterojunction, and sensitive detection of tumor suppressor gene P53 is realized.
"different concentrations of target and 10U of exonuclease III were added to the solution and incubated at 37 ℃ for 2h" was changed to "different concentrations of target and 10U of exonuclease III were added to the solution and incubated at 37 ℃ for 2.5h. "other conditions of preparation are the same as example 1, and a biosensing platform with the similar appearance and properties to example 1 is obtained. The results of P53 detection were the same as in example 1.
Example 4 use of DNA hydrogel and Bi 2 Sn 2 O 7 /Bi 2 S 3 A photoelectrochemical biosensor is developed by a heterojunction, and sensitive detection of tumor suppressor gene P53 is realized.
The other conditions of the preparation of adding the product chain PD obtained at different target concentrations into 10 mu L of DNA hydrogel and incubating for 1h for opening the gel are changed into adding the product chain PD obtained at different target concentrations into 10 mu L of DNA hydrogel and incubating for 2h for opening the gel are the same as those of the example 1, and the biosensor with the appearance and the property similar to those of the example 1 is obtained. The results of P53 detection were the same as in example 1.
Example 5 example 1 use of DNA hydrogel and Bi 2 Sn 2 O 7 /Bi 2 S 3 A photoelectrochemical biosensor is developed by a heterojunction, and sensitive detection of tumor suppressor gene P53 is realized.
And dropping 10 mu L of gel supernatant onto the substrate bismuth stannate material, and keeping the substrate bismuth stannate material wet at 37 ℃ for 10min to complete the anion exchange reaction. Instead, 10. Mu.L of the gel supernatant was dropped onto the substrate bismuth stannate material and kept wet at 37 ℃ for 20min to complete the anion exchange reaction. "other conditions of preparation are the same as example 1, and a biosensor having similar morphology and properties to example 1 is obtained. The results of P53 detection were the same as in example 1.
Claims (1)
1. By using Bi 2 Sn 2 O 7 /Bi 2 S 3 The application of the heterojunction sensitization photoinduced electrochemical biosensor in the detection of the P53 gene is characterized in that: the tumor suppressor gene P53 target initiates an exonuclease-assisted cyclic amplification reaction to release a large number of product chains; meanwhile, DNA hydrogel with target response is used for coating sulfide ions, the DNA hydrogel is opened through a product chain to quantitatively release the sulfide ions, and the sulfide ions and a substrate material Bi 2 Sn 2 O 7 Anion exchange reaction to form Bi 2 Sn 2 O 7 /Bi 2 S 3 A heterojunction; with pure Bi 2 Sn 2 O 7 Compared with the prior art, the heterojunction enables the amplification multiple of the photoelectric signal to reach 63 times, so that the ultra-sensitive detection of the target P53 gene is realized; in addition, the heterojunction is constructed, the defects that the single semiconductor photon-generated carrier is high in recombination speed and low in light energy utilization rate are overcome, and the polarity of photocurrent is converted from a cathode to an anode, so that the interference of background signals is eliminated, and the detection performance of the sensor is further enhanced;
the details are as follows
Step 1, preparation of DNA hydrogel: add 2. Mu.L 100. Mu.M acrylamide-modified DNA strand A and DNA strand B to 94. Mu.L stock solution; the stock solution contained 10mM Tris,1mM EDTA,140mM NaCl,5mM KCl,1mM CaCl 2 1mM MgCl 2 And 3% by mass of acrylamide; with N 2 Deoxidizing the solution for 15min, and then adding 1.5 mu L of freshly prepared initiator ammonium persulfate and 3 mu L of accelerator TEMED to react for 5min to generate polyacrylamide chains P-A and P-B; then 2. Mu.L of 100. Mu.M ligated DNA and sodium sulfide at a final concentration of 50mM were added to form a DNA hydrogel;
step 2, a target cyclic amplification process: mu.L of 1. Mu.M PD DNA and 10. Mu.L SiO 2 incubating-CP at 37 deg.C for 2h to obtain SiO 2 Adding targets with different concentrations and 10U of exonuclease III into the solution, incubating at 37 ℃ for 2h to obtain a large number of product chains PD, then incubating at 70 ℃ for 30min to inactivate the exonuclease III, centrifuging, and taking the supernatant at 4 ℃ for later use;
step 3.Bi 2 Sn 2 O 7 /Bi 2 S 3 Heterojunction electrochemical biosensors and their applications:ultrasonically dispersing 0.5mg of bismuth stannate powder into 1mL of secondary water containing 0.5% of chitosan, dripping 10 mu L of bismuth stannate solution into an ITO electrode, wherein the control area of the electrode is 0.16cm 2 Drying at room temperature to obtain a base material; then adding the obtained product chain PD into 10 mu L of DNA hydrogel, incubating for 1h, opening the gel and releasing sulfide ions; then 10 mu L of supernatant is dripped on a substrate bismuth stannate material, and the substrate bismuth stannate material is kept wet for 10min at 37 ℃ to complete anion exchange reaction to form Bi 2 Sn 2 O 7 /Bi 2 S 3 And heterojunction, a photoelectrochemical biosensor is constructed, and analysis and detection of the target P53 gene are realized.
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