CN113406160B - Photoelectrochemical biosensor and application thereof in methyltransferase activity detection - Google Patents
Photoelectrochemical biosensor and application thereof in methyltransferase activity detection Download PDFInfo
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Abstract
The invention discloses a photoelectrochemical biosensor and application thereof in methyltransferase activity detection, comprising a sensing electrode, gold nanoparticles, double-stranded DNA, endonuclease and dye, wherein the surface of the sensing electrode is covered with a P-type covalent organic polymer film; a recognition site is arranged in the double-stranded DNA, the recognition site 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, the other single-stranded DNA of the double-stranded DNA is arranged for connecting gold nanoparticles, and endonuclease is used for melting unmethylated double-stranded DNA; the dye is capable of intercalating into the phosphate backbone of double stranded DNA. The photoelectrochemical biosensor provided by the invention has the advantages of high detection sensitivity, quick operation and the like, and can sensitively and quickly detect the activity of methyltransferase.
Description
Technical Field
The invention belongs to the technical field of photoelectrochemistry detection, and relates to a photoelectrochemistry biosensor and application thereof in methyltransferase activity detection.
Background
The disclosure of this background section is only intended to increase the 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 already known to those of ordinary skill in the art.
Abnormal DNA methylation patterns depend on changes in DNA methyltransferase activity, which can serve as therapeutic targets and biomarkers for a variety of cancers and genetic diseases. Some diseases in humans are often associated with abnormal DNA methylation. Thus, DNA methylation analysis plays a vital role in early diagnosis of genetic diseases. To date, various methods for determining the DNA methylation and the DNA methyltransferase (DNA MTase) activity have been developed, such as high performance liquid chromatography, fluorescence, electrochemiluminescence, polymerase chain reaction, colorimetric assays, gel electrophoresis, and the like. However, most methods for determining the MTase activity of DNA have the disadvantages of expensive equipment, complicated operation process, and the need of specialized technicians. Compared with the method, the photoelectrochemical biosensor has the advantages of low cost, simple equipment, high sensitivity, quick operation and the like, and gradually attracts attention.
Photoelectrochemical (PEC) detection is an emerging, dynamically evolving analytical technique that has received great attention for its advantages in both electrochemical and optical analysis. Compared with the traditional electrochemical method and optical method, the PEC biosensor has the potential of higher sensitivity and low background signal due to separation of an excitation source and a detection signal. The signaling mechanisms of current PEC detection are mainly limited to changing electron donor/acceptor concentrations or changing diffusion efficiencies 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 unrepeatability and instability of the photoactive material on the electrode severely impedes charge transfer while the detection sensitivity is low, thereby affecting the application of PEC sensing in detecting DNA methyltransferase activity.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the photoelectrochemical biosensor and the application thereof in the detection of the methyltransferase activity, and the photoelectrochemical biosensor provided by the invention has the advantages of high detection sensitivity, quick operation and the like.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in one aspect, a photoelectrochemical biosensor includes:
a sensing electrode, the surface of which is covered with a P-type covalent organic polymer film;
gold nanoparticles and double-stranded DNA, wherein a recognition site is arranged in the double-stranded DNA, the recognition site can be methylated by methyltransferase, one single-stranded DNA of the double-stranded DNA is arranged for being connected with the P-type covalent organic polymer film, and the other single-stranded DNA of the double-stranded DNA is arranged for being connected with the gold nanoparticles;
an endonuclease for melting unmethylated double-stranded DNA;
a dye capable of intercalating 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 properties, and is beneficial to the sensing of photoelectric signals; meanwhile, strong pi-pi accumulation is formed between the P-type covalent organic polymer layers, so that high porosity and crystallinity are generated, the transport of charge carriers is facilitated, and a transport channel is provided for the transport of the carriers.
The surface of the gold nanoparticle can generate Surface Plasmon Resonance (SPR), and has the characteristics of visible light-induced charge separation, nearby strong local electric field and unique plasma light absorption. By surface plasmon resonance, not only can be used as a light collecting antenna of dye, but also the photocurrent transmission efficiency can be improved. Through the cooperation of the SPR effect and the dye sensitization, the detection sensitivity of the sensor can be greatly improved, and then the high-efficiency signal output is carried out through the P-type covalent organic polymer film, so that the high-sensitivity detection is realized.
The double-stranded DNA is internally provided with a recognition site capable of methylation of methyltransferase, whether the recognition site is methylated or not is used for judging the activity of methyltransferase, whether the methylation is also the key of endonuclease to double-stranded DNA melting, gold nanoparticles and dye can be combined only when the double-stranded DNA exists and are connected with a P-type covalent organic polymer film, and the synergy of SPR effect and dye sensitization is generated, so that the detection sensitivity can be improved, and the detection stability and reproducibility are enhanced.
Compared with other structures, the P-type covalent organic polymer film has thinner thickness, can lead the electrode to have better photoelectrochemical property and rich active sites, is not only beneficial to the receiving and the transmission of signals, but also beneficial to the connection of connecting groups so as to better connect double-stranded DNA, thereby improving the sensitivity, the reproducibility and the stability of detection.
In another aspect, the use of a photoelectrochemical biosensor as described above in the detection of methyltransferase activity.
In a third aspect, a method for detecting methyltransferase activity provides the photoelectrochemical biosensor described above;
mixing double-stranded DNA with a solution containing methyltransferase to be detected for the first time, adding endonuclease for the second time, adding a sensing electrode, gold nanoparticles and dye for mixed reaction, and carrying out photoelectrochemical detection on the sensing electrode after the reaction.
In a fourth aspect, the use of a photoelectrochemical biosensor as described above for screening for a methyltransferase agonist and/or a methyltransferase inhibitor, as the methyltransferase agonist and methyltransferase inhibitor are capable of affecting methyltransferase activity.
In a fifth aspect, a kit for detecting methyltransferase activity includes the photoelectrochemical biosensor described above, and a buffer solution.
The beneficial effects of the invention are as follows:
1. the invention adopts the P-type covalent organic polymer film to cover the electrode surface, has good cathode PEC performance and abundant active sites, and is favorable for the construction of the high-efficiency sensing of the photoelectrochemical biosensor.
2. The invention utilizes the SPR effect of AuNPs and the sensitization effect of RhB, the coupling of the two amplification effects can greatly amplify the photocurrent of the PEC biosensor, the sensitivity of the sensor is greatly improved, and the detection line is 0.022 units per milliliter. Highly specific recognition of enzymes may further enhance the specificity of the invention.
3. The cathode PEC biosensor designed by the invention can be widely 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 research and development and disease diagnosis.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram (A) of in-situ synthesis of COP film on transparent indium tin oxide coated glass (ITO) for PEC biosensor preparation schematic diagram (B) for M.SssIMTase activity detection in an embodiment of the invention;
FIG. 2 is a diagram showing the characterization of materials used in the examples of the present invention, wherein A is an infrared spectrogram of tris (4-aminophenyl) amine (TAPA) (1. Red line), 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) (2. Blue line), and COP (3. Black line); b is the XRD pattern of COP; c is an ultraviolet Diffuse Reflection (DRS) spectrogram of COP, wherein an internal illustration of C is a Tauc-Plot image of COP; XPS spectra of N1s and C1s, D and E being COP respectively; f is the ultraviolet-visible absorption spectrum of AuNPs; g and H are SEM top and cross-sectional views, respectively, of COP grown on ITO; i is a TEM image of AuNPs;
FIG. 3 shows photocurrent of different modified electrodes in an embodiment of the present invention, wherein FIG. A shows PEC performance studies of different modified electrodes, a is COP/ITO, b is AuNPs/COP/ITO, a and b are scanned in 0.1 mol/liter of phosphoric acid buffer solution (pH=7.4), c is AuNPs/COP/ITO in N 2 Scanning in saturated 0.1 mol per liter of phosphate buffer solution (ph=7.4), d is COP/ITO, e is AuNPs/COP/ITO, d, e are both scanned in 0.1 mol per liter of phosphate buffer solution (ph=7.4) containing 0.5 μg per ml of RhB; panel B is a feasibility study of PEC biosensors. Photocurrent responses of dsDNA/streptavidin/COP/ITO electrodes in the presence of hpaii+aunps (a), hpaii+aunps+rhb (b), hpaii+m.sssimtae+aunps (c), and hpaii+m.sssimtae+aunps+rhb (d), respectively;
FIG. 4 is a photocurrent generation mechanism of the COP of the present invention under visible light irradiation;
FIG. 5 is a graph showing the characteristics of sensitivity, selectivity and stability of the test results in the embodiment of the present invention, wherein A is the change of the photoelectric response intensity of M.SssIMTase with different concentrations (the concentrations from a to j are 0,0.05,0.1,0.5,1,5, 10, 20, 50 and 100 units per milliliter in sequence), and B is the linear relation between the photoelectric response intensity and the logarithm of the concentration of M.SssIMTase. C is the intensity of photoelectric response induced by HhaIMTase per milliliter, dam MTase per milliliter of 100 units and M.SssIMTase per milliliter of 100 units, and error bars represent standard deviations of three independent experiments; (D) The cathodic PEC biosensor continuously scans the PEC response curve for 14 cycles in 0.1 moles per liter of phosphate buffer (ph=7.4).
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. 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 present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the problems of poor reproducibility, instability, low sensitivity and the like in the existing photoelectric sensor for detecting the activity of methyltransferase, the invention provides a photoelectrochemical biosensor and application thereof in the detection of methyltransferase activity.
In an exemplary embodiment of the present invention, there is provided a photoelectrochemical biosensor including:
a sensing electrode, the surface of which is covered with a P-type covalent organic polymer film;
gold nanoparticles and double-stranded DNA, wherein a recognition site is arranged in the double-stranded DNA, the recognition site can be methylated by methyltransferase, one single-stranded DNA of the double-stranded DNA is arranged for being connected with the P-type covalent organic polymer film, and the other single-stranded DNA of the double-stranded DNA is arranged for being connected with the gold nanoparticles;
an endonuclease for melting unmethylated double-stranded DNA;
a dye capable of intercalating into the phosphate backbone of double stranded DNA.
The invention utilizes the cooperation of gold nanoparticle SPR effect and dye sensitization to improve the detection sensitivity of the sensor, and then carries out high-efficiency signal output through the P-type covalent organic polymer film, thereby realizing high-sensitivity detection. By providing double-stranded DNA of a recognition site capable of methylation by methyltransferase and by the cooperation of endonuclease, the cooperation of gold nanoparticle SPR effect and dye sensitization is realized, so that not only can the detection sensitivity be improved, but also the stability and reproducibility of photoelectrochemistry for detecting methyltransferase activity are enhanced.
In some examples of this embodiment, a single strand of double stranded DNA is attached to the P-type covalent organic polymer film by the coordination of biotin and streptavidin. In one or more embodiments, the surface of the P-type covalent organic polymer film is provided with streptavidin.
In some examples of this embodiment, another single strand of double stranded DNA is attached to the gold nanoparticle by a gold-sulphur bond.
In some examples of this embodiment, the P-type covalent organic polymer film and the gold nanoparticle are located at both ends of the double-stranded DNA, respectively.
In some examples of this embodiment, double stranded DNA is formed by hybridization of single stranded DNA1 with single stranded DNA 2;
the sequence of single-stranded DNA1 is: CAC CTC CGG ACT G;
the sequence of single-stranded DNA2 is: CAG TCC GGA GGT G.
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 schiff base reaction. The schiff base reaction is a reaction in which an aldehyde group reacts with a primary amine group to form a carbon-nitrogen double bond.
In one or more embodiments, the method of making the sensing electrode is: 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde solution and tris (4-aminophenyl) amine solution are added dropwise to the surface of the electrode to react. 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 reproducibility are improved.
In one or more embodiments, the 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde 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 invention, the application of the photoelectrochemical biosensor in the activity detection of methyltransferase is provided. The use is preferably for the diagnosis and treatment of non-diseases.
In a third embodiment of the present invention, a method for detecting methyltransferase activity is provided, and the photoelectrochemical biosensor is provided;
mixing double-stranded DNA with a solution containing methyltransferase to be detected for the first time, adding endonuclease for the second time, adding a sensing electrode, gold nanoparticles and dye for mixed reaction, and carrying out photoelectrochemical detection on the sensing electrode after the reaction.
The detection method is preferably aimed at diagnosis and treatment of non-diseases.
In some examples of this embodiment, the first incubation temperature is 36.5 to 37.5 ℃. The incubation time is 30-150 minutes.
In some examples of this embodiment, the second incubation temperature is 36.5 to 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, then gold nanoparticles are added dropwise for a fourth incubation, and then dye solution is added for a fifth incubation.
In a fourth embodiment of the present invention, there is provided the use of a photoelectrochemical biosensor as described above for screening for methyltransferase agonists and/or methyltransferase inhibitors.
In a fifth embodiment of the present invention, a kit for detecting methyltransferase activity is provided, which comprises the photoelectrochemical biosensor and a buffer solution.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Examples
Synthesis of COP film on ITO glass: the ITO glass was cut into 4.7 cm by 1cm pieces at 1.0 mol/liter NaOH, 10% H 2 O 2 And acetone, then thoroughly rinsed with ultra-pure water, and blow-dried with nitrogen prior to use. 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) and tris (4-aminophenyl) amine (TAPA) were dissolved in a mixed solution of mesitylene, ethanol and glacial acetic acid (volume ratio 5:5:1), respectively. And then uniformly mixing the DHNDA and TAPA solutions according to the volume ratio of 1:1, immediately dripping the mixed solution on the ITO surface, and reacting at room temperature in a closed system. The ITO glass was then immersed in methylene chloride to remove unreacted residual reagents, and the prepared electrode was dried at room temperature, at which time the ITO surface formed a uniform rose-brown film.
Synthesis of gold nanoparticles (AuNPs): 200 ml of 0.01% HAuCl 4 The solution was boiled under vigorous stirring, then 5 ml of 1% sodium citrate solution was added rapidly to the boiling solution. When the solution turned dark red, indicating the formation of AuNPs, the AuNPs solution was cooled with magnetic stirring.
Preparation of photoelectrochemical biosensor: the ITO surface was modified with a COP film. Subsequently, 20. Mu.l of a streptavidin solution (0.05 mg/ml) was dropped on the COP 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' with the sequence shown in SEQ ID NO.2.
The thiolated DNA-1 activation process 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 mM EDTA, 5 mM MgCl per liter) 2 10 millimoles per liter of Tris, pH=7.4) was incubated at 37℃for 30 minutes to obtain DNA-1/DNA-2 hybrid strands (dsDNA). The dsDNA probes were incubated in the reaction solution (160 μmol per liter SAM, 2.0 μL 1×netbuffer 2, and different concentrations of m.sssimtase) for 2 hours at 37 ℃. Subsequently, dsDNA probes were added to a1 XCutSmart buffer solution containing HpaII and incubated at 37℃for 2 hours, and the incubated reaction solution was dropped onto a streptavidin/COP/ITO electrode and incubated. After rinsing with PBS buffer solution, 20. Mu.l of AuNPs solution was added dropwise to the dsDNA/streptavidin/COP/ITO electrode and incubated for 8 hours, to obtain the AuNPs/dsDNA/streptavidin/COP/ITO electrode. The modified electrode was then incubated with 1 millimole per liter of RhB for 30 minutes.
The method comprises the steps of adopting a traditional three-electrode system, taking ITO glass with COP grown in situ as a working electrode, taking a platinum wire electrode as a counter electrode, taking an Ag/AgCl electrode as a reference electrode, taking PBS buffer solution as electrolyte solution under the irradiation of a xenon lamp, and detecting the change of photocurrent generated by a sensor.
1. Characterization of materials
The infrared spectrum of tris (4-aminophenyl) amine (TAPA) (FIG. 2A, 1) at 3338cm -1 And 1621cm -1 There is a stretching frequency corresponding to the N-H bond of the amino group and c=c of the benzene ring, respectively. The infrared spectrum of 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) (FIG. 2A, 2) was 2918cm -1 The aldehyde is characterized by stretching vibration at 1639cm -1 The characteristic telescopic vibration of carbonyl is provided. TAPA at 3338cm -1 The N-H peak and DHNDA peak at 1639cm -1 The characteristic stretching vibration at c=o disappeared after polymerization, and at 1603cm -1 And 2911cm -1 New characteristic peaks appear, corresponding to c=n and the stretching vibration peak of C-N on the benzene ring, respectively, confirming that the monomers of tris (4-aminophenyl) amine (TAPA) and 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) have been converted to COP (fig. 2a, 3). The XPS spectrum further demonstrated successful synthesis of COP (fig. 2E). High resolution at C1sIn X-ray photoelectron spectroscopy (XPS spectrum), a clear emission line was detected with a binding energy of 285.7eV due 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 by infrared spectroscopy, which further demonstrates the formation of imine bonds in the COP film. We further characterized COP films using X-ray diffraction (XRD). As shown in fig. 2B, the COP has two broad peaks at 11.5 ° and 21.8 °. Respectively corresponding to [100 ]]And [001]A plane. The broad peak at the higher 2θ angle (21.8 °) is caused by pi-pi stacking between COP layers.
The ultraviolet visible Diffuse Reflectance Spectrum (DRS) of COP films has a strong absorption band in the range of 300-600 nm with the tail extending even to 700 nm (fig. 2C). The band gap (E) of COP can be found by the following equation g )：
αhν＝A((hν-E g ) 1/2 (1)
Where α is the absorption coefficient, h is the Planck constant, v is the frequency of light, E g Band gap, A is a constant term. Tauc-Plot ((αhν) 2 vs (hν)) to the intersection with the transverse axis, the optical bandgap of COP was calculated to be 2.89eV.
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 diameter of AuNPs was about 13 nm and the particle size distribution was uniform. A top view image (fig. 2G) of a Scanning Electron Microscope (SEM) of COP shows that the ITO surface is covered with a continuous uniform film. The cross-sectional image of SEM hardly sees the boundary between film and ITO (fig. 2H), which demonstrates that the synthesized film is ultra-thin and has strong adhesion at the ITO surface.
2. Experimental verification of feasibility
To demonstrate the feasibility of this solution, the present example used a different method of modifying electrodes to prepare a cathodic PEC biosensor (fig. 3). The photocurrent (-2073 nanoamps, fig. 3A, curve b) of AuNPs/COP/ITO in 0.1 mol per liter of phosphate buffer solution (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 solution (ph=7.4). The increase in cathode photocurrent is mainly due to two reasons: (1) Direct contact of AuNPs with COP promotes charge transfer between them; (2) The SPR effect of AuNPs improves the separation efficiency of photogenerated carriers. The AuNPs/COP/ITO electrode was tested in a nitrogen deoxygenated 0.1 mol per liter phosphoric acid buffer solution (ph=7.4) and the photocurrent was reduced (fig. 3A, curve c), which illustrates that oxygen plays an important role as an electron acceptor in the generation of the cathode photocurrent. COP/ITO electrodes and AuNPs/COP/ITO electrodes were tested in 0.1 mol/L phosphate buffer solution (pH=7.4) containing 0.5. Mu.g/ml RhB, and their photocurrents were about-2512 nanoamperes (FIG. 3A, curve d) and-4201 nanoamperes (FIG. 3A, curve e), respectively, indicating that sensitization of RhB could enhance the photocurrent intensity of COP/ITO.
Fig. 3B shows PEC performance of PEC biosensors with different modified surfaces. It was found that dsDNA/streptavidin/COP/ITO photocurrent in the presence of hpaii+m.sssimtase+aunps (fig. 3B, curve c, -2048 nanoamps) was greater than that in the presence of hpaii+aunps alone (fig. 3B, curve a, -1120 nanoamps), demonstrating that this PEC biosensor was useful for detection of m.sssimtase. This is because the SPR effect of AuNPs improves the separation efficiency of photogenerated carriers, and the Conduction Band (CB) of COPs and AuNPs can react with dissolved oxygen to generate a significant cathode photocurrent. In addition, the photocurrent of the dsDNA/streptavidin/COP/ITO electrode in the presence of hpaii+m.sssimtase+aunps+rhb (fig. 3B, curve d, -2738 nanoamperes) was much higher than that in the presence of hpaii+m.sssimtase+aunps alone (fig. 3B, curve c, -2048 nanoamperes), indicating that dye RhB intercalation can increase photocurrent, further enhancing PEC biosensor signal.
Pec performance mechanism experiments
Fig. 4 depicts the mechanism by which a PEC biosensor constructed with COP films produces photocurrent under light conditions. Under irradiation of light, electrons in the COP Valence Band (VB) are transferred to the Conduction Band (CB), thereby generating electron-hole pairs. Electrons excited to the Conduction Band (CB) by the SPR enhancement effect can be rapidly injected to AuNPs. Subsequently, electrons on the AuNPs and COP Conduction Bands (CBs) react with dissolved oxygen in the electrolyte solution, producing a significant cathode photocurrent. RbB produces an excited state upon absorption of lightElectrons are transferred from the Valence Band (VB) of COP to RbB of the excited state (equation 2). Then, the electrons are transferred from the vat dye RhB- (formula 3) to O 2 Reduction product and RbB (equation 4) are produced. In addition, the SPR effect of AuNPs can be used as a light collecting antenna of RhB, so that the light collecting capacity is improved, and the photocurrent is enhanced.
RhB/COP+hv→RhB/COP (excitation) (2)
RhB*/COP→RhB - /COP(h + ) (hole injection) (3)
RhB - /COP(h + )+O 2 →RhB/COP(h + ) +reduction (4)
Product (dye regeneration and oxygen reduction)
4. Sensitivity experiment
In order to evaluate the sensitivity of the detection of m.sssimtase in the present technical solution, the present embodiment measures the relationship between the intensity of photoelectric response and the concentration of m.sssimtase under the optimal experimental conditions. As shown in fig. 5A, the intensity of the electrical response increases with increasing concentration of m.sssimtase. The log of the intensity of the photoelectric response and the concentration of M.SssIMTase has a good linear relationship in the range of 0.05-100 units per milliliter (FIG. 5B), and the linear correlation equation is I= -343.48lgC-1971.23 (R 2 = 0.9964), where I represents the photoelectric value and C represents the concentration of m.sssimtase (units per milliliter). The limit of detection was calculated to be 0.022 units per milliliter from the average value of the control 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 with Dam methylase (Dam MTase) and HhaI methylase (HhaI MTase) as interfering enzymes. As shown in FIG. 5C, the intensity of the photoelectric response of M.SssIMTase is much greater than that of Dam MTase and HhaIMTase, since Dam MTase and HhaIMTase cannot methylate CpG sites. Thus, the PEC biosensor developed has good specificity for m.sssimtase.
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 biosensor, with an intra-batch variation coefficient of 0.21% measured, indicating good reproducibility of the PEC biosensor. We further studied the stability of the proposed PEC cell sensor. Fig. 5D shows that the excitation light source repeats a switching lamp cycle every 20 seconds, with no significant change in light response intensity after 280 seconds of continuous irradiation. The Relative Standard Deviation (RSD) of the photo current values for 14 switching lamp cycles was 1.23%, indicating that the PEC biosensor has good stability.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Shandong university of teachers and students
<120> photoelectrochemical biosensor and application thereof in methyltransferase activity detection
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 13
<212> DNA
<213> artificial sequence
<400> 1
cacctccgga ctg 13
<210> 2
<211> 13
<212> DNA
<213> artificial sequence
<400> 2
cagtccggag gtg 13
Claims (10)
1. A photoelectrochemical biosensor for detecting methyltransferase activity, comprising:
a sensing electrode, the surface of which is covered with a P-type covalent organic polymer film;
gold nanoparticles and double-stranded DNA, wherein a recognition site is arranged in the double-stranded DNA, the recognition site can be methylated by methyltransferase, one single-stranded DNA of the double-stranded DNA is arranged for being connected with the P-type covalent organic polymer film, and the other single-stranded DNA of the double-stranded DNA is arranged for being connected with the gold nanoparticles;
an endonuclease for melting unmethylated double-stranded DNA;
a dye capable of intercalating into the phosphate backbone of double-stranded DNA;
double-stranded DNA is formed by hybridization of single-stranded DNA1 and single-stranded DNA 2;
the sequence of single-stranded DNA1 is: CAC CTC CGG ACT G;
the sequence of single-stranded DNA2 is: CAG TCC GGAGGT G;
the P-type covalent organic polymer film and the gold nanoparticles are respectively positioned at two ends of the double-stranded DNA;
only when double-stranded DNA exists, gold nanoparticles and dye can be combined and connected with a P-type covalent organic polymer film, so that the cooperation of SPR effect and dye sensitization is generated, the detection sensitivity can be improved, and the detection stability and reproducibility are enhanced.
2. The photoelectrochemical biosensor of claim 1, wherein a single strand of double stranded DNA is attached to the P-type covalent organic polymer membrane by biotin-streptavidin coordination; the surface of the P-type covalent organic polymer film is provided with streptavidin;
the other single-stranded DNA of the double-stranded DNA is linked to the gold nanoparticle by a gold-sulfur bond.
3. The photoelectrochemical biosensor of claim 1, wherein said P-type covalent organic polymer film is obtained from 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde and tris (4-aminophenyl) amine by schiff base reaction;
the preparation method of the sensing electrode comprises the following steps: 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde solution and tris (4-aminophenyl) amine solution are dropwise added to the surface of the electrode for reaction;
the 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde solution and the tris (4-aminophenyl) amine solution contain mesitylene, ethanol and acetic acid;
the electrode is conductive glass.
4. Use of a photoelectrochemical biosensor as claimed in any one of claims 1 to 3 in the detection of methyltransferase activity.
5. A method for detecting methyltransferase activity, characterized by providing the photoelectrochemical biosensor of any one of claims 1 to 3;
mixing double-stranded DNA with a solution containing methyltransferase to be detected for the first time, adding endonuclease for the second time, adding a sensing electrode, gold nanoparticles and dye for mixed reaction, and carrying out photoelectrochemical detection on the sensing electrode after the reaction.
6. The method for detecting methyltransferase activity according to claim 5, wherein the first incubation temperature is 36.5 to 37.5 ℃; the incubation time is 30-150 minutes.
7. The method for detecting methyltransferase activity according to claim 5, wherein the second incubation temperature is 36.5 to 37.5 ℃; the incubation time is 30-150 minutes.
8. The method for detecting methyltransferase activity according to claim 5, wherein the solution after the second reaction is dropped onto the sensor electrode for the third incubation, then gold nanoparticles are dropped for the fourth incubation, and then dye solution is added for the fifth incubation.
9. Use of a photoelectrochemical biosensor as claimed in any of claims 1 to 3 for screening for methyltransferase agonists and/or methyltransferase inhibitors.
10. A kit for detecting methyltransferase activity, which is characterized by comprising the photoelectrochemical biosensor as claimed in any one of claims 1 to 3 and a buffer solution.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105353006A (en) * | 2015-11-11 | 2016-02-24 | 安徽理工大学 | Photoelectric sensor and application thereof, and preparation method of working electrode |
| CN105784810A (en) * | 2016-04-20 | 2016-07-20 | 华东交通大学 | Label-free aptamer sensor and method for detecting bisphenol A |
| CN110702910A (en) * | 2019-08-27 | 2020-01-17 | 东南大学 | Photoelectrochemical immunosensor for detecting activity of DNA methylase and preparation method and application thereof |
| CN111830104A (en) * | 2020-06-05 | 2020-10-27 | 山东师范大学 | Photoelectrochemistry biosensor and preparation method and application thereof |
| CN111944873A (en) * | 2020-08-18 | 2020-11-17 | 山东师范大学 | Nano-gold biosensor for detecting DNA (deoxyribonucleic acid) methylation transferase as well as detection method and application thereof |
| CN112225926A (en) * | 2020-09-25 | 2021-01-15 | 山东大学 | Covalent organic material film and preparation method and application thereof |
| CN112226484A (en) * | 2020-10-27 | 2021-01-15 | 山东师范大学 | Fluorescent biosensor of beta-glucose transferase, detection method and application |
-
2021
- 2021-05-27 CN CN202110582957.1A patent/CN113406160B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105353006A (en) * | 2015-11-11 | 2016-02-24 | 安徽理工大学 | Photoelectric sensor and application thereof, and preparation method of working electrode |
| CN105784810A (en) * | 2016-04-20 | 2016-07-20 | 华东交通大学 | Label-free aptamer sensor and method for detecting bisphenol A |
| CN110702910A (en) * | 2019-08-27 | 2020-01-17 | 东南大学 | Photoelectrochemical immunosensor for detecting activity of DNA methylase and preparation method and application thereof |
| CN111830104A (en) * | 2020-06-05 | 2020-10-27 | 山东师范大学 | Photoelectrochemistry biosensor and preparation method and application thereof |
| CN111944873A (en) * | 2020-08-18 | 2020-11-17 | 山东师范大学 | Nano-gold biosensor for detecting DNA (deoxyribonucleic acid) methylation transferase as well as detection method and application thereof |
| CN112225926A (en) * | 2020-09-25 | 2021-01-15 | 山东大学 | Covalent organic material film and preparation method and application thereof |
| CN112226484A (en) * | 2020-10-27 | 2021-01-15 | 山东师范大学 | Fluorescent biosensor of beta-glucose transferase, detection method and application |
Non-Patent Citations (4)
| Title |
|---|
| A sensitive signal-on electrochemical assay for MTase activity using AuNPs amplification;Xiaoxiao He et al.;《Biosensors and Bioelectronics》;20110723;第28卷(第1期);第298至303页 * |
| Construction of a Dye-Sensitized and Gold Plasmon-Enhanced Cathodic Photoelectrochemical Biosensor for Methyltransferase Activity Assay;Lin Cui et al.;《Analytical Chemistry》;20210714;第93卷(第29期);全文 * |
| Development of electrochemical genosensor for MYCN oncogene detection using rhodamine B as electroactive label;Thalles Douglas Souza e Silva et al.;《Journal of Solid State Electrochemistry》;20160714;第20卷;第2411至2418页 * |
| Xiaoxiao He et al..A sensitive signal-on electrochemical assay for MTase activity using AuNPs amplification.《Biosensors and Bioelectronics》.2011,第28卷(第1期),298-303. * |
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