Photoelectrochemical biosensor and application thereof in methyl transferase activity detection
1. A photoelectrochemical biosensor, comprising:
the sensing electrode, the electrode surface covers the organic polymer membrane of P-type covalence;
the double-stranded DNA is internally provided with a recognition site which can be methylated by methyltransferase, one single-stranded DNA of the double-stranded DNA is arranged for connecting the P-type covalent organic polymer film, and the other single-stranded DNA of the double-stranded DNA is arranged for connecting the gold nanoparticles;
an endonuclease for melting unmethylated double-stranded DNA;
a dye capable of inserting into the phosphate backbone of double stranded DNA.
2. The photoelectrochemical biosensor of claim 1, wherein one single-stranded DNA of the double-stranded DNA is linked to the P-type covalent organic polymer film by the conjugation of biotin and streptavidin; preferably, streptavidin is arranged on the surface of the P-type covalent organic polymer membrane;
or, the other single-stranded DNA of the double-stranded DNA is connected with the gold nanoparticle through a gold-sulfur bond.
3. The photoelectrochemical biosensor of claim 1, wherein the P-type covalent organic polymer film and the gold nanoparticles are located at both ends of the double-stranded DNA, respectively.
4. The photoelectrochemical biosensor of claim 1, wherein the double-stranded DNA is formed by hybridization of single-stranded DNA1 with single-stranded DNA 2;
the sequence of the single-stranded DNA1 is: CAC CTC CGG ACT G, respectively;
the sequence of the single-stranded DNA2 is: CAG TCC GGA GGT G are provided.
5. The photoelectrochemical biosensor according to claim 1, wherein said P-type covalent organic polymer film is obtained by reacting 2, 6-dihydroxynaphthalene-1, 5-dicarbaldehyde with tris (4-aminophenyl) amine using a schiff base;
preferably, the preparation method of the sensing electrode comprises the following steps: dropwise adding a2, 6-dihydroxynaphthalene-1, 5-diformaldehyde solution and a tris (4-aminophenyl) amine solution to the surface of the electrode to react;
preferably, the 2, 6-dihydroxynaphthalene-1, 5-dimethaldehyde solution and the tris (4-aminophenyl) amine solution both contain mesitylene, ethanol and acetic acid;
preferably, the electrode is conductive glass.
6. Use of the photoelectrochemical biosensor of any one of claims 1 to 5 in the detection of methyltransferase activity.
7. A method for detecting a methyltransferase activity, which comprises providing the photoelectrochemical biosensor according to any one of claims 1 to 5;
mixing double-stranded DNA with a solution to be detected containing methyltransferase for the first incubation, adding incision enzyme for the second incubation, then adding a sensing electrode, gold nanoparticles and dye for mixed reaction, and carrying out photoelectrochemical detection on the reacted sensing electrode.
8. The method for detecting the activity of methyltransferase according to claim 7, wherein the first incubation temperature is 36.5 to 37.5 ℃; preferably, the incubation time is 30-150 minutes;
or the temperature of the second incubation is 36.5-37.5 ℃; preferably, the incubation time is 30-150 minutes;
or, dropwise adding the solution after the second reaction onto the sensing electrode for the third incubation, dropwise adding the gold nanoparticles for the fourth incubation, and then adding the dye solution for the fifth incubation.
9. Use of the photoelectrochemical biosensor of any one of claims 1 to 5 in screening for methyltransferase agonists and/or methyltransferase inhibitors.
10. A kit for detecting a methyltransferase activity, comprising the photoelectrochemical biosensor according to any one of claims 1 to 5 and a buffer solution.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Aberrant DNA methylation patterns depend on alterations in DNA methyltransferase activity, which may serve as therapeutic targets and biomarkers for various cancers and genetic diseases. Some diseases in humans are often associated with abnormal DNA methylation. Therefore, DNA methylation analysis has a crucial role for the early diagnosis of genetic diseases. To date, various methods for measuring DNA methylation and DNA methyltransferase (DNA MTase) activity have been developed, such as high performance liquid chromatography, fluorescence, electrochemiluminescence, polymerase chain reaction, colorimetric assay, gel electrophoresis, and the like. However, most of the methods for measuring the activity of DNA MTase have disadvantages of expensive equipment, complicated operation process, and the need for a skilled technician. Compared with the method, the photoelectrochemistry biosensor has the advantages of low cost, simple equipment, high sensitivity, quick operation and the like, and gradually draws attention of people.
Photoelectrochemical (PEC) detection is an emerging, dynamically evolving analytical technique that has received much attention because of its combined advantages of electrochemical and optical analysis. PEC biosensors have the potential for higher sensitivity and low background signal due to the separation of the excitation source and detection signal compared to traditional electrochemical and optical methods. Current signal transfer mechanisms of PEC detection are limited primarily to altering electron donor/acceptor concentrations or altering diffusion efficiency to achieve signal attenuation/enhancement. The drop coating method is still the electrode preparation method widely adopted at present. However, the inventors have found that the non-repeatability and instability of photoactive materials on electrodes severely hamper charge transport, while the detection sensitivity is low, thus affecting the use of PEC sensing for detecting DNA methyltransferase activity.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a photoelectrochemical biosensor and an application thereof in the detection of the activity of the methyltransferase.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in one aspect, a photoelectrochemical biosensor comprises:
the sensing electrode, the electrode surface covers the organic polymer membrane of P-type covalence;
the double-stranded DNA is internally provided with a recognition site which can be methylated by methyltransferase, one single-stranded DNA of the double-stranded DNA is arranged for connecting the P-type covalent organic polymer film, and the other single-stranded DNA of the double-stranded DNA is arranged for connecting the gold nanoparticles;
an endonuclease for melting unmethylated double-stranded DNA;
a dye capable of inserting into the phosphate backbone of double stranded DNA.
The P-type covalent organic polymer not only has the advantage of large specific surface area, but also has excellent photoelectric property, and is beneficial to sensing photoelectric signals; meanwhile, strong pi-pi accumulation formed between layers of the P-type covalent organic polymer can generate high porosity and crystallinity, which is beneficial to the transport of charge carriers and provides a transport channel for the transfer of the charge carriers.
The surface of the gold nanoparticle can generate Surface Plasmon Resonance (SPR) and has visible light induced charge separation, a nearby strong local electric field and unique plasma light absorption characteristics. Through surface plasmon resonance, the optical fiber can be used as a light collecting antenna of dye, and the photocurrent transmission efficiency can be improved. Through the cooperation of SPR effect and dye sensitization, the detection sensitivity of the sensor can be greatly improved, and high-efficiency signal output is carried out through the P-type covalent organic polymer membrane, so that high-sensitivity detection is realized.
The double-stranded DNA is internally provided with a recognition site capable of methylating the methyltransferase, whether the methylation of the recognition site is carried out or not is also the key for the unwinding of the methyltransferase by the endonuclease, and only when the double-stranded DNA exists, the gold nanoparticles and the dye can be combined and connected with a P-type covalent organic polymer membrane to generate the synergy of the SPR effect and the dye sensitization, so that the detection sensitivity can be improved, and the detection stability and the reproducibility are enhanced.
Compared with other structures, the P-type covalent organic polymer film has thinner thickness, can ensure that the electrode has better photoelectrochemical performance and rich active sites, is not only beneficial to receiving and transmitting signals, but also beneficial to connecting the connecting groups so as to better connect double-stranded DNA, thereby improving the sensitivity, reproducibility and stability of detection.
In another aspect, a use of the above-described photoelectrochemical biosensor in the detection of methyltransferase activity.
In a third aspect, a method for detecting the activity of methyltransferase is provided, which comprises providing the above-mentioned photoelectrochemical biosensor;
mixing double-stranded DNA with a solution to be detected containing methyltransferase for the first incubation, adding incision enzyme for the second incubation, then adding a sensing electrode, gold nanoparticles and dye for mixed reaction, and carrying out photoelectrochemical detection on the reacted sensing electrode.
Since methyltransferase agonists and methyltransferase inhibitors can affect the activity of methyltransferases, in a fourth aspect, a use of the above-described photoelectrochemical biosensor for screening for methyltransferase agonists and/or methyltransferase inhibitors.
In a fifth aspect, a kit for detecting the activity of methyltransferase comprises the above photoelectrochemical biosensor and a buffer solution.
The invention has the beneficial effects that:
1. the invention adopts the P-type covalent organic polymer film to cover the surface of the electrode, has good cathode PEC performance and abundant active sites, and is beneficial to the construction of high-efficiency sensing of the photoelectrochemical biosensor.
2. The invention utilizes SPR effect of AuNPs and sensitization of RhB, the coupling of the two amplification effects can greatly amplify photocurrent of the PEC biosensor, the sensitivity of the sensor is greatly improved, and the detection line is 0.022 unit per milliliter. The high specificity recognition of the enzyme may further enhance the specificity of the invention.
3. The cathode PEC biosensor designed by the invention can be applied to detection of other DNA methyltransferases and DNA modification enzymes only by changing the recognition sequence, and has wide application prospects in the fields of drug development and disease diagnosis.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
Fig. 1 is a schematic diagram of in situ synthesis of COP film on transparent indium tin oxide coated glass (ITO) for example of the invention (a), a schematic diagram of PEC biosensor preparation for m.ssimtase activity detection (B);
FIG. 2 is a representation of the materials used in the examples of the present invention, A is an infrared spectrum of tris (4-aminophenyl) amine (TAPA) (1. red line), 2, 6-dihydroxynaphthalene-1, 5-dicarbaldehyde (DHNDA) (2. blue line), COP (3. black line); b is an XRD image of COP; c is an ultraviolet Diffuse Reflectance (DRS) spectrogram of COP, wherein an internal inset of C is a Tauc-Plot image of COP; XPS spectra of N1s and C1s with D and E being COPs, respectively; f is the ultraviolet-visible absorption spectrum of AuNPs; g and H are SEM top view and cross-sectional view, respectively, of COP grown on ITO; i is a TEM image of AuNPs;
FIG. 3 shows the photocurrent of different modified electrodes in the example of the present invention, where A is PEC performance study of different modified electrodes, a is COP/ITO, b is AuNPs/COP/ITO, a and b are both scanned in 0.1 mol/L phosphoric acid buffer solution (pH 7.4), and c is AuNPs/COP/ITO in N2Scanning was performed in saturated 0.1 mol per liter phosphate buffer (pH 7.4), d is COP/ITO, e is AuNPs/COP/ITO, d, e were both scanned in 0.1 mol per liter phosphate buffer (pH 7.4) containing 0.5 micrograms per milliliter of RhB; panel B is a feasibility study of the PEC biosensor. Photocurrent responses of the dsDNA/streptavidin/COP/ITO electrode in the presence of HpaII + AuNPs (a), HpaII + AuNPs + RhB (b), HpaII + M.SssIMTase + AuNPs (c) and HpaII + M.SssIMTase + AuNPs + RhB (d), respectively;
FIG. 4 is a photocurrent generation mechanism of COP under visible light irradiation according to an embodiment of the present invention;
fig. 5 is a graph representing the results of the sensitivity, selectivity and stability tests performed in the examples of the present invention, where a is the variation of the intensity of the photo-electric response of m.ss imtase at different concentrations (concentration: 0, 0.05, 0.1, 0.5, 1,5, 10, 20, 50 and 100 units per ml in order from a to j), and B is the linear relationship between the intensity of the photo-electric response and the logarithm of the m.ss imtase concentration. C is the photoelectric response intensity induced by hhamitase in units per ml, Dam MTase in 100 units per ml and m.sssmimtase in 100 units per ml, error bars represent the standard deviation of three independent experiments; (D) the cathode PEC biosensor continuously scanned 14 cycles of PEC response curves in 0.1 mol/l phosphate buffer solution (pH 7.4).
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In view of the problems of poor reproducibility, instability, low sensitivity and the like of the conventional photoelectric sensor for detecting the activity of the methyltransferase, the invention provides a photoelectric chemical biosensor and application thereof in the detection of the activity of the methyltransferase.
In an exemplary embodiment of the present invention, there is provided a photoelectrochemical biosensor including:
the sensing electrode, the electrode surface covers the organic polymer membrane of P-type covalence;
the double-stranded DNA is internally provided with a recognition site which can be methylated by methyltransferase, one single-stranded DNA of the double-stranded DNA is arranged for connecting the P-type covalent organic polymer film, and the other single-stranded DNA of the double-stranded DNA is arranged for connecting the gold nanoparticles;
an endonuclease for melting unmethylated double-stranded DNA;
a dye capable of inserting into the phosphate backbone of double stranded DNA.
The invention utilizes the cooperation of the SPR effect of the gold nanoparticles and the dye sensitization to improve the detection sensitivity of the sensor, and then the high-efficiency signal output is carried out through the P-type covalent organic polymer film, thereby realizing the high-sensitivity detection. By providing double-stranded DNA of a recognition site capable of methylation of methyltransferase and matching with endonuclease, the coordination of the SPR effect of gold nanoparticles and the dye sensitization effect is realized, the detection sensitivity can be improved, and the stability and the reproducibility of the activity of the photoelectrochemical detection methyltransferase are enhanced.
In some examples of this embodiment, one single-stranded DNA of the double-stranded DNA is linked to the P-type covalent organic polymer membrane by biotin-streptavidin conjugation. In one or more embodiments, streptavidin is disposed on the surface of the P-type covalent organic polymer membrane.
In some embodiments of this embodiment, the other single-stranded DNA of the double-stranded DNA is linked to the gold nanoparticle by a gold-sulfur bond.
In some embodiments of this embodiment, the P-type covalent organic polymer membrane and the gold nanoparticles are located at each end of the double-stranded DNA.
In some embodiments of this embodiment, the double-stranded DNA is formed by hybridization of single-stranded DNA1 with single-stranded DNA 2;
the sequence of the single-stranded DNA1 is: CAC CTC CGG ACT G, respectively;
the sequence of the single-stranded DNA2 is: CAG TCC GGA GGT G are provided.
In some examples of this embodiment, the P-type covalent organic polymer film is obtained from 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde and tris (4-aminophenyl) amine by a schiff base reaction. The Schiff base reaction is a reaction that aldehyde group reacts with primary amine group to form carbon-nitrogen double bond.
In one or more embodiments, the sensing electrode is prepared by: and dropwise adding a2, 6-dihydroxynaphthalene-1, 5-diformaldehyde solution and a tris (4-aminophenyl) amine solution to the surface of the electrode for reaction. The P-type covalent organic polymer film is generated on the surface of the electrode in situ, so that the signal transmission is facilitated, and the stability and the reproducibility are improved.
In one or more embodiments, the 2, 6-dihydroxynaphthalene-1, 5-dimethaldehyde solution and the tris (4-aminophenyl) amine solution each contain mesitylene, ethanol, and acetic acid.
In one or more embodiments, the electrode is a conductive glass. The conductive glass is preferably ITO.
In another embodiment of the present invention, there is provided a use of the above-mentioned photoelectrochemical biosensor in the detection of methyltransferase activity. The use is preferably for non-disease diagnosis and treatment purposes.
In a third embodiment of the present invention, there is provided a method for detecting a methyltransferase activity, comprising providing the above-mentioned photoelectrochemical biosensor;
mixing double-stranded DNA with a solution to be detected containing methyltransferase for the first incubation, adding incision enzyme for the second incubation, then adding a sensing electrode, gold nanoparticles and dye for mixed reaction, and carrying out photoelectrochemical detection on the reacted sensing electrode.
The detection method is preferably aimed at diagnosis and treatment of non-diseases.
In some embodiments of this embodiment, the first incubation temperature is 36.5-37.5 ℃. The incubation time is 30-150 minutes.
In some embodiments of this embodiment, the second incubation temperature is 36.5-37.5 ℃. The incubation time is 30-150 minutes.
In some examples of this embodiment, the solution after the second reaction is added dropwise to the sensing electrode for a third incubation, and then gold nanoparticles are added dropwise for a fourth incubation, and then a dye solution is added for a fifth incubation.
In a fourth embodiment of the present invention, there is provided a use of the above-mentioned photoelectrochemical biosensor in screening for a methyltransferase agonist and/or a methyltransferase inhibitor.
In a fifth embodiment of the present invention, a kit for detecting methyltransferase activity is provided, which includes the above-mentioned photoelectrochemical biosensor and a buffer solution.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Examples
Synthesis of COP film on ITO glass: ITO glass was cut into 4.7 cm by 1cm pieces at 1.0 mol/L NaOH, 10% H2O2And sonicated in acetone, then thoroughly rinsed with ultra pure water and blown dry with nitrogen before use. Respectively dissolving 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) and tris (4-aminophenyl) amine (TAPA) in a mixed solution of mesitylene, ethanol and glacial acetic acid (in a volume ratio of 5:5: 1). Then, the DHNDA solution and the TAPA solution are uniformly mixed according to the volume ratio of 1:1, and then the mixed solution is immediately dropped on the ITO surface for reaction in a closed system at room temperature. The ITO glass was then soaked in dichloromethane to remove unreacted residual reagents and the prepared electrode was dried at room temperature, whereupon a uniform rose-brown film formed on the ITO surface.
Synthesis of gold nanoparticles (AuNPs): 200 ml of 0.01% HAuCl4The solution was boiled under vigorous stirring, and then 5 ml of 1% sodium citrate solution was added rapidly to the boiling solution. When the solution turned deep red, indicating the formation of AuNPs, the AuNPs solution was cooled while maintaining magnetic stirring.
Preparing a photoelectrochemical biosensor: and modifying the COP film on the ITO surface. Then, 20. mu.l of streptavidin solution (0.05 mg/ml) was dropped on the COP thin film-modified electrode, and dried to obtain a streptavidin/COP/ITO-modified electrode.
The sequence of DNA-1 from 5 'to 3' is: 5 '-CAC CTC CGG ACT G-SH-3' with the sequence shown in SEQ ID NO. 1.
The sequence of DNA-2 from 5 'to 3' is: 5 '-CAG TCC GGA GGT G-biotin-3', the sequence is shown in SEQ ID NO. 2.
The activation process of thiolated DNA-1 is: the disulfide-bonded oligonucleotides were reduced with tris (2-carboxyethyl) phosphine hydrochloride (TCEP) for one hour.
DNA-1 and DNA-2 in hybridization buffer (1.0 mmole/L EDTA, 5 mmole/L MgCl)210 mmoles per liter Tris, pH 7.4) at 37 ℃ for 30 minutes to obtain DNA-1/DNA-2 hybrid strands (dsDNA). The dsDNA probe was incubated in the reaction solution (160. mu. mol/L SAM, 2.0. mu.L 1 XNEBuffer 2 and different concentrations of M.SssIMTase) for 2 hours at 37 ℃. The dsDNA probe was then added to a1 XCutSmart buffer containing HpaII, incubated at 37 ℃ for 2 hours, and the incubated reaction solution was incubated dropwise on a streptavidin/COP/ITO electrode. After washing with PBS buffer solution, 20 microliter of AuNPs solution is dripped on a dsDNA/streptavidin/COP/ITO electrode for incubation for 8 hours to obtain the AuNPs/dsDNA/streptavidin/COP/ITO electrode. The modified electrode was then incubated with 1 mmol/l RhB for 30 minutes.
A traditional three-electrode system is adopted, ITO glass with COP grown in situ is used as a working electrode, a platinum wire electrode is used as a counter electrode, an Ag/AgCl electrode is used as a reference electrode, and under the irradiation of a xenon lamp, PBS buffer solution is used as electrolyte solution to detect the change of photocurrent generated by a sensor.
1. Characterization of materials
Infrared spectrum (FIG. 2A, 1) of tris (4-aminophenyl) amine (TAPA) at 3338cm-1And 1621cm-1The stretching frequency corresponds to the N-H bond of the amino group and the C ═ C of the benzene ring. Infrared spectrum (FIGS. 2A, 2) of 2, 6-dihydroxynaphthalene-1, 5-dicarbaldehyde (DHNDA) at 2918cm-1Characteristic stretching vibration of aldehyde at 1639cm-1Characteristic stretching vibration of carbonyl group. TAPA 3338cm-1The stretching peak at N-H and DHNDA are 1639cm-1The characteristic stretching vibration of C ═ O disappears after polymerization, but it is 1603cm-1And 2911cm-1New characteristic peaks appear, corresponding to the stretching vibration peaks of C ═ N and C-N on the benzene ring, respectively, and the preparation of tris (4-aminophenyl) amine (TAPA) and 2, 6-dihydroxynaphthalene-1, 5-dicarbaldehyde (DHNDA) is confirmedThe monomer had been converted to COP (fig. 2A, 3). The XPS spectra further demonstrate the successful synthesis of COP (fig. 2E). In the high resolution X-ray photoelectron spectrum (XPS spectrum) of C1s, a clear emission line was detected with a binding energy of 285.7eV, which is attributed to the C ═ N bond. C ═ N at 398.8eV was also observed in the XPS spectrum of N1s (fig. 2D). These results are consistent with those observed with infrared spectroscopy, which further confirms the formation of imine bonds in COP films. We further characterized COP films using X-ray diffraction (XRD). As shown in fig. 2B, COP has two broad peaks at 11.5 ° and 21.8 °. Respectively correspond to [100 ]]And [001 ]]And (4) a plane. The broad peak at the higher 2 theta angle (21.8 deg.) is caused by pi-pi stacking between COP layers.
The ultraviolet-visible Diffuse Reflection Spectrum (DRS) of the COP film has a strong absorption band in the range of 300-600 nm, and the absorption tail even extends to 700 nm (FIG. 2C). The band gap (E) of COP can be obtained by the following equationg)：
αhν＝A((hν-Eg)1/2 (1)
Where α is the absorption coefficient, h is the Planck constant, v is the frequency of the light, EgIs the band gap and a is a constant term. Matching Tauc-Plot ((alpha h v)2vs (h ν)) is extrapolated to the intersection with the horizontal axis, and the optical bandgap of COP is calculated to be 2.89 eV.
An absorption peak was observed at 520 nm for 13 nm AuNPs (fig. 2F). And as can be seen from TEM images of AuNPs (fig. 2I), the AuNPs have a diameter of about 13 nm and a uniform particle size distribution. A top view image (fig. 2G) of a Scanning Electron Microscope (SEM) of COP shows the ITO surface covered with a continuous uniform thin film. The boundary between the thin film and the ITO was hardly seen in the cross-sectional image of SEM (fig. 2H), which confirmed that the resultant thin film was ultra-thin and had strong adhesion on the ITO surface.
2. Experimental validation of feasibility
To demonstrate the feasibility of this approach, this example used a method of preparing a cathodic PEC biosensor using a different modified electrode (fig. 3). The photocurrent (-2073 nanoamps, fig. 3A, curve b) of AuNPs/COP/ITO in 0.1 mol per liter of phosphate buffer (pH 7.4) was higher than the photocurrent (-819 nanoamps, fig. 3A, curve a) of COP/ITO in 0.1 mol per liter of phosphate buffer (pH 7.4). The increase in cathode photocurrent is primarily due to two reasons: (1) direct contact of AuNPs with COPs facilitates charge transfer between them; (2) the SPR effect of AuNPs improves the separation efficiency of photogenerated carriers. The decrease in photocurrent was measured with AuNPs/COP/ITO electrodes in 0.1 mol/l phosphate buffer (pH 7.4) deoxygenated with nitrogen (fig. 3A, curve c), indicating that oxygen as an electron acceptor plays an important role in the generation of cathode photocurrent. The photocurrent of the COP/ITO electrode and the AuNPs/COP/ITO electrode, respectively, was measured in 0.1 mol/l phosphate buffer solution (pH 7.4) containing 0.5 μ g/ml RhB at about-2512 nanoamps (fig. 3A, curve d) and-4201 nanoamps (fig. 3A, curve e), indicating that the sensitization by RhB can enhance the photocurrent intensity of COP/ITO.
Fig. 3B shows the PEC performance of the PEC biosensors of the different modified surfaces. The discovery that the photocurrent of dsDNA/streptavidin/COP/ITO in the presence of hpai ii + m.ssimtase + AuNPs (fig. 3B, curve c, -2048 nanoamps) was greater than the photocurrent in the presence of hpai ii + AuNPs alone (fig. 3B, curve a, -1120 nanoamps) demonstrates that the PEC biosensor can be used for m.ssimtase detection. The reason is that the separation efficiency of photogenerated carriers is improved by the SPR effect of AuNPs, and the Conduction Band (CB) of COPs and AuNPs can react with dissolved oxygen to generate obvious cathode photocurrent. Furthermore, the photocurrent of the dsDNA/streptavidin/COP/ITO electrode in the presence of hpai ii + m.sssimtase + AuNPs + RhB (fig. 3B, curve d, -2738 nanoamps) was much higher than the photocurrent in the presence of hpai ii + m.ssimtase + AuNPs alone (fig. 3B, curve c, -2048 nanoamps), indicating that the intercalation of the dye RhB can increase the photocurrent, further enhancing the signal of the PEC biosensor.
PEC Performance mechanistic experiments
Fig. 4 depicts the mechanism by which PEC biosensors constructed with COP films produce photocurrent under light conditions. Under light irradiation, electrons in the COP Valence Band (VB) are transferred to the Conduction Band (CB), thereby generating electron-hole pairs. Due to the SPR enhancement effect, electrons excited on the Conduction Band (CB) by the COP can be rapidly injected into AuNPs. Subsequently, electrons on AuNPs and COP Conduction Band (CB) willReacts with dissolved oxygen in the electrolyte solution to produce a significant cathode photocurrent. RbB, upon absorption of light, produce RbB in the excited state, and the electrons are transferred from the Valence Band (VB) of the COP to RbB in the excited state (equation 2). Then, the electron is transferred from the vat dye RhB- (equation 3) to O2To produce a reduced product RbB (equation 4). In addition, the SPR effect of AuNPs can be used as a light collecting antenna of RhB, so that the light collecting capability is improved, and the enhancement of photocurrent is realized.
RhB/COP + hv → RhB/COP (excited) (2)
RhB*/COP→RhB-/COP(h+) (hole injection) (3)
RhB-/COP(h+)+O2→RhB/COP(h+) + reduction (4)
Products (regeneration of dyes and reduction of oxygen)
4. Sensitivity test
In order to evaluate the sensitivity of detecting m.sssimtase in the present technical solution, under the optimal experimental conditions, the present example measured the relationship between the photoelectric response intensity and the m.sssimtase concentration. As shown in fig. 5A, the electrical response intensity increased with increasing m.ss imtase concentration. The logarithm of the photoelectric response intensity and the M.SssIMTase concentration has a good linear relation in the range of 0.05-100 units per milliliter (figure 5B), and the linear correlation equation is that I is-343.48 lgC-1971.23(R is R20.9964) where I represents the photocurrent value and C represents the concentration of m.ss imtase (units per ml). The limit of detection was calculated as 0.022 units per ml based on the mean of the controls plus three times the standard deviation.
5. Specificity, reproducibility and stability experiments
To investigate the selectivity of PEC biosensors, we evaluated the selectivity of the constructed PEC biosensors using Dam methylase (Dam MTase) and hhal methylase (HhaIMTase) as interfering enzymes. As shown in fig. 5C, the photo-response intensity of m.ssstimtase was much greater than that of Dam MTase and hhamitase, because Dam MTase and hhamitase failed to methylate CpG sites. Therefore, the PEC biosensor developed has good specificity for m.sssimtase.
In order to evaluate the reproducibility of the proposed PEC biosensor, three different PEC biosensors were prepared under the same conditions to investigate the intra-assay accuracy of the PEC biosensors, with an intra-batch coefficient of variation of 0.21% of the assay, indicating that the PEC biosensors have good reproducibility. We further investigated the stability of the proposed PEC cell sensor. Fig. 5D shows that the excitation light source repeats the on-off cycle every 20 seconds, with no significant change in the light response intensity after 280 seconds of continuous illumination. The Relative Standard Deviation (RSD) of the photocurrent values for 14 switching lamp cycles was 1.23%, indicating good stability of the PEC biosensor.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
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