CN111690721B - Photo-induced electrochemical biosensor and preparation method and application thereof - Google Patents
Photo-induced electrochemical biosensor and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a photoinduced electrochemical biosensor based on DNA nanometer branch amplification signals and a preparation method and application thereof; the technical scheme of the invention is that trace DNA is utilized to initiate exonuclease III to assist in cyclic amplification reaction, so that a large number of product chains are generated, DNA rolling ring amplification and chain hybridization are combined to form a nano branch, a large number of manganese porphyrins are embedded into the nano branch to quench bismuth sulfide photoelectric signals, a simple, convenient and practical Photoelectrochemical (PEC) sensor is prepared, ultra-sensitive detection of the trace DNA is realized, and the research has good application potential in the fields of clinical diagnosis, gene therapy, environmental monitoring, food safety and the like.
Description
Technical Field
The invention relates to the technical field of sensors, in particular to a photo-induced electrochemical biosensor and a preparation method and application thereof.
Background
There is an increasing need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances. What is needed most urgently is a method for measuring small quantities of drugs, metabolites, microorganisms and other substances of diagnostic value. Such as narcotics, poisons, drugs for therapeutic purposes, hormones, pathogenic microorganisms, viruses, antibodies, metabolites, enzymes, nucleic acids, etc., the detection of specific trace DNA sequences is very critical in the fields of clinical diagnosis, gene therapy, environmental monitoring, food safety, etc.
Photoelectrochemical (PEC) biosensing is a new detection method developed by combining Photoelectrochemical analysis technology and biosensing technology. The detection principle is to determine the concentration of the detection object based on the photoelectric conversion characteristics of the photoelectric active substance. In the photoelectrochemical detection process, light is used as an excitation signal to excite the photosensitive substance, and an electrical signal is used as a detection signal. The excitation signal is in a different form than the detection signal, resulting in a reduced background signal and, therefore, a higher sensitivity of the PEC analysis technique.
However, the photoelectric signal and sensitivity of the current photoelectric sensor are limited, and a large number of researches show that manganese porphyrin is a metal complex integrating manganese into porphyrin, and has the advantages of low production cost, stable chemical performance, high catalytic performance, high efficiency, excellent biocompatibility and the like, so that the manganese porphyrin can be used for photoelectrochemical analysis.
According to the invention, DNA rolling circle amplification and chain hybridization are combined to form a nano branch, and a branch porphyrin manganese quenches a bismuth sulfide photoelectric signal to prepare a sensitive, simple and practical photoelectrochemical platform for detecting trace target DNA.
Disclosure of Invention
In view of the above, the present invention provides a photo-induced electrochemical biosensor based on a novel photoelectric material and DNA nano-tree amplified signals; and a preparation method of the photo-induced electrochemical biosensor and an analysis application of the photo-induced electrochemical biosensor for detecting trace target DNA.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of making a photo-electrochemical biosensor, comprising:
forming DNA nanometer tree branches through rolling ring amplification technology and chain type hybridization, and embedding porphyrin manganese into the nanometer tree branches to realize bismuth sulfide (Bi) 2 S 3 ) Quenching of photoelectric signals, a simple, convenient and practical photoinduced electrochemical sensor is prepared, and ultra-sensitive double-signal detection of target DNA is realized.
Preferably, the method for preparing the photo-electrochemical biosensor comprises the following steps:
(1) Preparing a DNA rolling circle template, mixing a circular template sequence DNA, a connecting DNA, secondary water and a buffer solution of 10 XT 4DNA ligase, annealing, naturally cooling to room temperature to obtain a mixed solution, adding the T4DNA ligase into the mixed solution for connection, performing primary inactivation treatment on the T4DNA ligase in the mixed solution, adding an exonuclease system, performing cutting treatment, and performing secondary inactivation treatment on exonuclease I and exonuclease III in the mixed solution to obtain the DNA rolling circle template for later use
(2) An exonuclease III assisted circulation amplification process: annealing HP, naturally cooling to room temperature to obtain a hairpin structure, and then carrying out mixed reaction with target DNA, exonuclease III and exonuclease buffer solution to obtain a product chain;
(3) The PEC platform preparation process of porphyrin manganese quenching bismuth sulfide comprises the following steps: first, bi is added 2 S 3 Dropping the suspension on the surface of an ITO electrode, dropping AuNPs after the electrode is dried, adding H1 solution after the electrode is dried, adding an MCH (hydrogen-doped carbon nitride) sealing plate, dropping a product chain on the surface of the electrode for primary incubation treatment, adding phi29DNA polymerase, 10 XPhi 29DNA polymerase buffer solution, dNTPs and a DNA rolling ring template on the surface of the electrode for secondary incubation treatment, adding H2 solution and H3 solution for tertiary incubation treatment, adding manganese porphyrin for fourth incubation treatment, washing the modified electrode with TE buffer solution, and drying in the air to obtain the sensor.
Preferably, the preparation method specifically comprises the following steps:
(1) Preparing a DNA rolling circle template: mixing 1 mu L of ring template sequence DNA, 3 mu L of connecting DNA, 4 mu L of secondary water and 3 mu L of buffer solution of 10 XT 4DNA ligase, annealing, naturally cooling to room temperature to obtain mixed solution, adding 110-130U of T4DNA ligase into the mixed solution for connection, then carrying out primary inactivation treatment on the T4DNA ligase in the mixed solution, then adding 4 mu L of exonuclease buffer solution, 10-30U of exonuclease I and 90-110U of exonuclease III, carrying out cutting treatment, then carrying out secondary inactivation treatment on the exonuclease I and the exonuclease III in the mixed solution to obtain a DNA rolling ring template, and storing the obtained DNA rolling ring template at 2-6 ℃ for later use;
(2) An exonuclease III assisted circulation amplification process: annealing 18 mu L of HP, naturally cooling to room temperature to obtain a hairpin structure, and then carrying out mixed reaction with 18 mu L of target DNA, 20-40U of exonuclease III and 4 mu L of exonuclease buffer solution to obtain a Product Chain (PC);
(3) The PEC platform preparation process of porphyrin manganese quenching bismuth sulfide comprises the following steps: first, 15. Mu.L of Bi was added 2 S 3 Dropping the suspension on the surface of an ITO electrode, dropping 15 mu L of AuNPs after the electrode is dried, adding 15 mu L of H1 solution after the electrode is dried, reacting for 11-13H at 4-25 ℃, adding an MCH (multi-channel metal-oxide-semiconductor) closing plate, washing by using TE (transverse moving bed) buffer solution to remove unconnected H1, dropping 10 mu L of product chains on the surface of the electrode for first incubation treatment, washing by using TE buffer solution to remove redundant PC, adding 0.4 mu L of phi29DNA polymerase, 1 mu L of phi29DNA polymerase buffer solution, 2 mu L of dNTPs and 6.6 mu L of DNA rolling ring template for second incubation treatment, washing by using TE buffer solution, adding 5 mu L of H2 solution and 5 mu L of H3 solution for third incubation treatment, adding 10 mu L of manganese porphyrin for fourth incubation treatment, finally washing the modified electrode by using TE buffer solution, and drying in the air to obtain the sensor.
Preferably, the DNA sequence of the loop template sequence is:
TCGATCTCAGATCCTAAGCCGCACCCAAAGACTG；
the connecting DNA sequence is:
GATCGACAGTCT；
the HP sequence is as follows:
TCTCAGATGGATTCGGCGTGAAT*T*T*T*TTCACGCCGAATCCATC TGAGAGGCCGTCTATGCGTGAACTG
the H1 sequence is as follows:
TCTGACAGCTAGAGTCTAGGATTCGGCGTGGGTTTTTCACGCCGAA TCCATCTGAGATTTTTTT-SH；
the H2 sequence is as follows:
AACCCACGCCGAATGGGGGGATTCGGCG；
the H3 sequence is ATTCGGCGTGGGTTCGCCGAATCCCCCCCC.
Preferably, in the step (1), the concentration of the circle template sequence DNA is 1. Mu.M; the concentration of the ligated DNA was 100. Mu.M.
Preferably, in the step (1), the annealing temperature is 93-97 ℃ and the annealing time is 4-6min;
the temperature of the connection is 35-37 ℃, and the time is 1-2h;
the temperature of the first inactivation treatment is 62-68 ℃, and the time is 8-12min;
the cutting temperature is 35-37 ℃, and the cutting time is 1-2h;
the temperature of the second inactivation treatment is 77-83 ℃, and the time is 12-18min.
Preferably, in the step (2), the concentration of the HP is 1 μ M;
the temperature of the annealing treatment is 93-97 ℃, and the time is 4-6min;
the temperature of the mixing reaction is 35-37 ℃, and the time is 1-3h.
Preferably, in the step (3), bi is contained 2 S 3 The concentration of the suspension is 2mg/mL; the concentration of the H1 solution is 1 mu M; the concentration of the H2 solution is 1 mu M; the concentration of the H3 solution is 1 mu M; the concentration of the porphyrin manganese is 100 mu M, wherein H1, H2 and H3 are required to be annealed into hairpin structures, disulfide bonds are opened through reduction, the annealing temperature is 93-97 ℃, and the annealing time is 4-6min.
Preferably, in the step (3), the temperature of the first incubation treatment is 35-37 ℃, and the incubation time is 1.5-2.5h;
the temperature of the second incubation treatment is 35-37 ℃, and the time is 2.5-3.5h;
the temperature of the third incubation treatment is 35-37 ℃, and the time is 1.5-2.5h;
the temperature of the fourth incubation treatment is 35-37 ℃, and the time is 1.5-2.5h.
Further, the photo-induced electrochemical biosensor is prepared by the preparation method.
Furthermore, the photo-induced electrochemical biosensor is used for realizing sensitive detection of trace target DNA.
In the invention, the bismuth sulfide nanorod is used as a signal probe to detect trace DNA with ultra-sensitivity. Bismuth sulfide is used as a substrate to combine with the gold nanoparticles to generate a high-strength photoelectric signal. The target cyclic amplification process is to utilize exonuclease III to generate a large number of product chains for opening DNA hairpins fixed on a substrate, combine rolling ring amplification and chain hybridization to form a DNA nano branch rich in double chains, load a large number of quenching molecular porphyrin manganese, effectively quench a bismuth sulfide signal, and realize ultrasensitive photoelectric detection of target trace DNA according to the change of a quenched photoelectric signal.
Through the technical scheme, compared with the prior art, the invention has the following beneficial effects:
the invention designs a novel dendritic DNA nano structure, a large amount of porphyrin manganese is embedded, and Bi is obviously quenched 2 S 3 The photoelectric signal of the photosensitive material realizes the high-sensitivity detection of the target DNA and has great innovation on the development of photoelectric sensing. The RCA and HCR combined double amplification technology can form a large number of DNA double-stranded dendritic structures, thereby greatly amplifying PEC signals and improving the detection sensitivity. The work opens up a new direction for PEC detection using DNA nanostructures to amplify signals, and is expected to be a simple and practical PEC biosensing technology for gene therapy and disease diagnosis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of a photoelectrochemical biosensor based on DNA nanotree amplification signals.
FIG. 2 electrophoretic characterization during preparation of example 1: the method comprises the following steps of (A) an ExoIII assisted circulation amplification process, (B) a DNA chain hybridization process, and (C) a DNA conversion amplification process.
FIG. 3 TEM image of example 1 during preparation: (A) TEM image of DNA rolling-up amplified product, (B) TEM image of DNA chain-hybridization product, and (C) TEM image of DNA dendron (rolling-up amplified product combined with chain-hybridization).
FIG. 4 (A) shows photocurrent signal response curves of the photo-electric signal sensor prepared in example 1 at different target concentrations of DNA, i.e., 100nM, 10nM, 1.0nM, 100pM, 10pM, 1.0pM, 100fM, 10fM (from a to h), and (B) shows linear relationships between photocurrent changes and target concentrations.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
Mixing 1 mu L of padnock DNA with the concentration of 100 mu M, 3 mu L of assistant DNA with the concentration of 100 mu M, 4 mu L of secondary water and 3 mu L of buffer solution of 10 XT 4DNA ligase, annealing at 95 ℃ for 5min, naturally cooling to room temperature, adding 120U of T4DNA ligase into the mixed solution, connecting at 37 ℃ for 1h, reacting the mixed solution at 65 ℃ for 10min to inactivate the T4DNA ligase, cooling to room temperature, adding 4 mu L of exonuclease buffer solution, 20U of exonuclease I and 100U of exonuclease III, cutting at 37 ℃ for 2h, reacting the mixed solution at 80 ℃ for 15min for inactivating the exonuclease I and the exonuclease III, cooling to room temperature to obtain a DNA rolling ring template, and storing the obtained DNA rolling ring template at 4 ℃ for later use.
mu.L of HP at a concentration of 1. Mu.M was annealed at 95 ℃ for 5min, then naturally cooled to room temperature to obtain hairpin structures, and then 18. Mu.L of HP at a concentration of 1. Mu.M, 18. Mu.L of target DNA at various concentrations, 20U of exonuclease III, and 4. Mu.L of exonuclease buffer were mixed and reacted at 37 ℃ for 2h to obtain a large number of product strands (PC).
Adding Bi 2 S 3 The suspension (15. Mu.L, 2 mg/mL) was dropped on the ITO electrode surface (control electrode area 0.16 cm) 2 ) After drying in air, auNPs solution (15. Mu.L) was dropped on the surface of the electrode, after drying the electrode, H1 solution (15. Mu.L, 1. Mu.M) was dropped on the surface of the electrode, reaction was carried out at 4 ℃ for 12 hours, then unbound H1 was removed by washing with TE buffer solution, MCH seal plate was added, then cyclic amplification product PC (10. Mu.L) was dropped thereon and incubated at 37 ℃ for 2 hours, excess PC was removed by washing with TE buffer solution, 0.4. Mu.L of phi29DNA polymerase, 1. Mu.L of phi29DNA polymerase buffer solution, 2. Mu.L of dNTPs and 6.6. Mu.L of DNA rolling circle template were added to the electrode surface and incubated at 37 ℃ for 3 hours, washing with TE buffer solution was carried out, then H2 solution (5. Mu.L, 1. Mu.M), H3 solution (5. Mu.L, 1. Mu.M) was added at 37 ℃ for 2 hours, manganese porphyrin (10. Mu.L, 100. Mu.M) was added at 37 ℃ for 2 hours, finally the modified electrode was incubated with TE buffer solution and then dried, and PEC was used for detection.
Example 2
Mixing 1 mu L of padnock DNA with the concentration of 100 mu M, 3 mu L of assistant DNA with the concentration of 100 mu M, 4 mu L of secondary water and 3 mu L of buffer solution of 10 XT 4DNA ligase, annealing at 97 ℃ for 4min, naturally cooling to room temperature, adding 130U of T4DNA ligase into the mixed solution, connecting at 37 ℃ for 2h, reacting the mixed solution at 68 ℃ for 8min to inactivate the T4DNA ligase, cooling to room temperature, adding 4 mu L of exonuclease buffer solution, 30U of exonuclease I and 110U of exonuclease III, cutting at 37 ℃ for 2h, reacting the mixed solution at 83 ℃ for 12min for inactivating the exonuclease I and the exonuclease III, cooling to room temperature to obtain a DNA rolling ring template, and storing the obtained DNA rolling ring template at 6 ℃ for later use.
mu.L of HP at a concentration of 1. Mu.M was annealed at 97 ℃ for 4min, then naturally cooled to room temperature to obtain hairpin structures, and then 18. Mu.L of HP at a concentration of 1. Mu.M, 18. Mu.L of target DNA at various concentrations, 40U of exonuclease III, and 4. Mu.L of exonuclease buffer were mixed and reacted at 37 ℃ for 1h to obtain a large number of product strands (PC).
Adding Bi 2 S 3 The suspension (15. Mu.L, 2 mg/mL) was dropped on the ITO electrode surface (control electrode area 0.16 cm) 2 ) After drying in air, auNPs solution (15. Mu.L) was dropped on the surface of the electrode, after drying the electrode, H1 solution (15. Mu.L, 1. Mu.M) was dropped on the surface of the electrode, reaction was carried out at 25 ℃ for 11 hours, then unattached H1 was removed by washing with TE buffer solution, MCH seal plate was added, then cycle amplification product PC (10. Mu.L) was dropped thereon and incubated at 37 ℃ for 1.5 hours, excess PC was removed by washing with TE buffer solution, 0.4. Mu.L of phi29DNA polymerase, 1. Mu.L of phi29DNA polymerase buffer solution, 2. Mu.L of dNTPs and 6.6. Mu.L of DNA rolling circle template were added to the electrode surface and incubated at 37 ℃ for 2.5 hours, washing with TE buffer solution, then H2 solution (5. Mu.L, 1. Mu.M), H3 solution (5. Mu.L, 1. Mu.M) was added and incubated at 37 ℃ for 1.5 hours, manganese porphyrin (10. Mu.L, 100. Mu.M) was added and then modified at 37 ℃ for 1.5 hours, finally the electrode was incubated with TE buffer solution and then dried and then PEC, and then the electrode was washed and then examined with TE buffer solution was used for detection.
Example 3
Mixing 1 mu L of padlocDNA with the concentration of 100 mu M, 3 mu L of assistant DNA with the concentration of 100 mu M, 4 mu L of secondary water and 3 mu L of buffer solution of 10 XT 4DNA ligase, annealing for 6min at 93 ℃, naturally cooling to room temperature, adding 1110U of T4DNA ligase into the mixed solution, connecting for 1h at 35 ℃, reacting the mixed solution at 62 ℃ for 12min to inactivate the T4DNA ligase, cooling to room temperature, adding 4 mu L of exonuclease buffer solution, 10U of exonuclease I and 90U of exonuclease III, cutting for 1h at 35 ℃, reacting the mixed solution at 77 ℃ for 18min for inactivating the exonuclease I and the exonuclease III, cooling to room temperature to obtain a DNA rolling ring template, and storing the obtained DNA rolling ring template at 2 ℃ for later use.
mu.L of HP with concentration of 1. Mu.M was annealed at 93 ℃ for 6min, then naturally cooled to room temperature to obtain hairpin structures, and then 18. Mu.L of HP with concentration of 1. Mu.M, 18. Mu.L of target DNA with different concentrations, 20U of exonuclease III and 4. Mu.L of exonuclease buffer solution were mixed and reacted at 35 ℃ for 3h to obtain a large number of product strands (PC).
Adding Bi 2 S 3 The suspension (15. Mu.L, 2 mg/mL) was dropped on the ITO electrodePolar surface (control electrode area 0.16 cm) 2 ) After drying in air, auNPs solution (15. Mu.L) was dropped on the surface of the electrode, after drying the electrode, H1 solution (15. Mu.L, 1. Mu.M) was dropped on the surface of the electrode, reaction was carried out at 4 ℃ for 13 hours, then unattached H1 was removed by washing with TE buffer solution, MCH seal plate was added, then cycle amplification product PC (10. Mu.L) was dropped thereon and incubated at 35 ℃ for 2.5 hours, excess PC was removed by washing with TE buffer solution, 0.4. Mu.L of phi29DNA polymerase, 1. Mu.L of phi29DNA polymerase buffer solution, 2. Mu.L of dNTPs and 6.6. Mu.L of DNA rolling circle template were added to the electrode surface and incubated at 35 ℃ for 3.5 hours, washing with TE buffer solution, then H2 solution (5. Mu.L, 1. Mu.M), H3 solution (5. Mu.L, 1. Mu.M) was added and incubated at 35 ℃ for 2.5 hours, manganese porphyrin (10. Mu.L, 100. Mu.M) was added and then incubated at 35 ℃ for 2.5 hours, finally the electrode was washed with TE buffer solution and then dried and then the PEC was used for detection.
The target DNA sequences used in examples 1-3 were:
CAGTTC ACGCATAGACGG*C*C*
all the used H1, H2 and H3 need to be annealed to form a hairpin structure, a disulfide bond is opened through reduction, and the annealing parameters are consistent with the HP annealing parameters.
Sensitive photoelectrochemical detection
Under optimal reaction conditions, the PEC detection response of the biosensor to the target DNA concentration was studied. As shown in FIG. 4A, the PEC signal gradually decreased with the target DNA concentration from 10fM to 100nM (curves a-h). As can be seen in FIG. 4B, the change in PEC signal is well linear with the target DNA concentration. The inset is a corrected graph of the detected target DNA, illustrating the successful sensor preparation.
In the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A preparation method of a photo-induced electrochemical biosensor is characterized by comprising the following steps:
(1) Preparing a DNA rolling ring template, mixing a ring template sequence DNA, a connecting DNA, secondary water and a buffer solution of 10 XT 4DNA ligase, annealing, naturally cooling to room temperature to obtain a mixed solution, adding the T4DNA ligase into the mixed solution for connection, performing primary inactivation treatment on the T4DNA ligase in the mixed solution, adding an exonuclease system, performing cutting treatment, and performing secondary inactivation treatment on exonuclease I and exonuclease III in the mixed solution to obtain the DNA rolling ring template for later use;
(2) An exonuclease III assisted circulation amplification process: annealing HP, naturally cooling to room temperature to obtain a hairpin structure, and then carrying out mixed reaction with target DNA, exonuclease III and exonuclease buffer solution to obtain a product chain;
(3) The PEC platform preparation process of porphyrin manganese quenching bismuth sulfide comprises the following steps: first, bi is added 2 S 3 Dropping the suspension on the surface of an ITO electrode, dropping AuNPs after the electrode is dried, adding H1 solution after the electrode is dried, adding an MCH sealing plate, dropping a product chain on the surface of the electrode for primary incubation treatment, adding phi29DNA polymerase, 10 XPi 29DNA polymerase buffer solution, dNTPs and a DNA rolling ring template on the surface of the electrode for secondary incubation treatment, adding H2 solution and H3 solution for tertiary incubation treatment, adding manganoporphyrin for fourth incubation treatment, washing the modified electrode with TE buffer solution, and drying in the air to obtain the sensing electrodeA device;
wherein the secondary water is water subjected to twice distillation;
the DNA sequence of the ring template sequence is as follows:
TCGATCTCAGATCCTAAGCCGCACCCAAAGACTG；
the connecting DNA sequence is:
GATCGACAGTCT；
the HP sequence is:
TCTCAGATGGATTCGGCGTGAATTTTTTCACGCCGAATCCATCTGAGAGGCCGTCTATGCGTGAACTG
the H1 sequence is as follows:
TCTGACAGCTAGAGTCTAGGATTCGGCGTGGGTTTTTCACGCCGAATCCATCTGAGATTTTTTT-SH；
the H2 sequence is as follows:
AACCCACGCCGAATGGGGGGATTCGGCG；
the H3 sequence is ATTCGGCGTGGGTTCGCCGAATCCCCCCCC.
2. The method for preparing the photo-induced electrochemical biosensor according to claim 1, comprising the following steps:
(1) Preparing a DNA rolling circle template: mixing 1 mu L of ring template sequence DNA, 3 mu L of connecting DNA, 4 mu L of secondary water and 3 mu L of buffer solution of 10 XT 4DNA ligase, annealing, naturally cooling to room temperature to obtain mixed solution, adding 110-130U of T4DNA ligase into the mixed solution, connecting for 1 hour at 37 ℃, performing first inactivation treatment on the T4DNA ligase in the mixed solution, adding 4 mu L of exonuclease buffer solution, 10-30U of exonuclease I and 90-110U of exonuclease III, cutting for 2 hours at 37 ℃, performing second inactivation treatment on the exonuclease I and the exonuclease III in the mixed solution to obtain a DNA rolling ring template, and storing the obtained DNA rolling ring template at 2-6 ℃ for later use;
(2) An exonuclease III assisted circulation amplification process: annealing 18 mu L of HP, naturally cooling to room temperature to obtain a hairpin structure, and then carrying out mixed reaction with 18 mu L of target DNA, 20-40U of exonuclease III and 4 mu L of exonuclease buffer solution to obtain a product chain;
(3) The PEC platform preparation process of porphyrin manganese quenching bismuth sulfide comprises the following steps: first, 15. Mu.L of Bi was added 2 S 3 Dropping the suspension on the surface of an ITO electrode, dropping 15 mu L of AuNPs after the electrode is dried, adding 15 mu L of H1 solution after the electrode is dried, reacting for 11-13H at 4-25 ℃, adding an MCH (multi-channel metal-oxygen) closing plate, dropping 10 mu L of product chains on the surface of the electrode for primary incubation treatment, adding 0.4 mu L of phi29DNA polymerase, 1 mu L of 10 XPhi 29DNA polymerase buffer solution, 2 mu L of dNTPs and 6.6 mu L of DNA rolling ring template on the surface of the electrode for secondary incubation treatment, adding 5 mu L of H2 solution and 5 mu L of H3 solution for tertiary incubation treatment, adding 10 mu L of manganoporphyrin for fourth incubation treatment, finally washing the modified electrode by using TE buffer solution, and drying in the air to obtain the sensor.
3. The method for preparing a photo-induced electrochemical biosensor according to claim 2, wherein in the step (1), the concentration of the ring template sequence is 1 μ M; the concentration of the ligated DNA was 100. Mu.M.
4. The method for preparing the photo-induced electrochemical biosensor according to claim 2, wherein in the step (1), the annealing temperature is 93-97 ℃ and the annealing time is 4-6min;
the temperature of the connection is 35-37 ℃, and the time is 1-2h;
the temperature of the first inactivation treatment is 62-68 ℃, and the time is 8-12min;
the cutting temperature is 35-37 ℃, and the cutting time is 1-2h;
the temperature of the second inactivation treatment is 77-83 deg.C, and the time is 12-18min.
5. The method for preparing a photo-electrochemical biosensor as claimed in claim 2, wherein in step (2), the concentration of HP is 1 μ M;
the temperature of the annealing treatment is 93-97 ℃, and the time is 4-6min;
the temperature of the mixing reaction is 35-37 ℃, and the time is 1-3h.
6. The method for preparing the photo-induced electrochemical biosensor according to claim 2, wherein in the step (3), the Bi is 2 S 3 The concentration of the suspension is 2mg/mL; the concentration of the H1 solution is 1 mu M; the concentration of the H2 solution is 1 mu M; the concentration of the H3 solution is 1 mu M; the concentration of the manganese porphyrin is 100 mu M.
7. The method for preparing the photo-induced electrochemical biosensor as claimed in claim 2, wherein in the step (3), the temperature of the first incubation treatment is 35-37 ℃, and the incubation time is 1.5-2.5h;
the temperature of the second incubation treatment is 35-37 ℃, and the time is 2.5-3.5h;
the temperature of the third incubation treatment is 35-37 ℃, and the time is 1.5-2.5h;
the temperature of the fourth incubation treatment is 35-37 ℃, and the time is 1.5-2.5h.
8. A photo-electrochemical biosensor prepared according to the method of any one of claims 1-7.
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