CN109946360B - Sensor and method for detecting 8-hydroxyguanine DNA glycosidase - Google Patents
Sensor and method for detecting 8-hydroxyguanine DNA glycosidase Download PDFInfo
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Abstract
The invention discloses a sensor and a method for detecting 8-hydroxyguanine DNA glycosidase, wherein the sensor comprises a first electrode for generating an electric signal for detecting 8-hydroxyguanine DNA glycosidase, and the surface of the first electrode is modified with CP-DNA and TD-DNA combined by base complementary pairing; the detection method comprises the following steps: modifying CP-DNA to the surface of the first electrode; the TD-DNA and the CP-DNA are complementarily paired and then combined to the surface of a first electrode, after obtaining a ferrocene electric signal, the first electrode is immersed into buffer solution containing hOGG1 with different concentrations to carry out 8-hydroxyguanine cutting and hybridization chain reaction, and an electric signal containing hexaammonium ruthenium is obtained; and reading the differential pulse voltammetric signal on the first electrode after soaking, and calculating the relationship between the reduction amount of the ferrocene electrical signal, the increase amount of the hexa-ammonium ruthenium electrical signal and the concentration of hOGG 1. The invention combines the hybridization chain reaction with the biosensor, greatly reduces the detection limit of the concentration of the substance to be detected hOGG1, and has the advantages of obvious specificity, higher stability, high sensitivity, rapidness and low cost.
Description
Technical Field
The invention relates to the technical field of DNA glycosidase detection, in particular to a sensor and a method for detecting 8-hydroxyguanine DNA glycosidase.
Background
8-oxoguanine (8-oxoG) is an important oxidative damage to DNA and is the major product formed by oxygen radicals attacking DNA or RNA. 8-oxoG can cause base transversions during DNA replication, and can activate protooncogenes or inhibit the activity of cancer suppressor genes.
hOGG1 is a single-base excision repair enzyme, has activity of glycosidase and AP lyase, and can specifically recognize excision of 8-oxoG to repair oxidative damage of DNA. The change of the activity of hOGG1 can obviously influence the ability of cell to repair 8-oxoG, individual tumor susceptibility and the sensitivity to chemotherapeutic drugs, and the detection of the activity of hOGG1 can provide important basis for the relationship between a DNA repair mechanism and tumors. Therefore, the realization of the rapid and sensitive detection of hOGG1 is of great significance.
Currently, the existing methods for detecting the activity of hOGG1 mainly include a radioisotope method, a High Performance Liquid Chromatography (HPLC), a reverse transcriptase-polymerase chain reaction (RT-PCR) and an endonuclease cleavage method. However, the above method has problems such as time and labor consuming, low sensitivity, complicated operation, etc.
Disclosure of Invention
Aiming at the defects in the technology, the invention provides the sensor for detecting the 8-hydroxyguanine DNA glycosidase, which greatly reduces the detection limit of the concentration of the substance hOGG1 to be detected.
The invention also provides a method for detecting the sensor for detecting the 8-hydroxyguanine DNA glycosidase, which combines the hybridization chain reaction with the biosensor and has the advantages of obvious specificity, higher stability, high sensitivity, rapidness and low cost.
To achieve these objects and other advantages in accordance with the present invention, the present invention is implemented by the following solutions:
the sensor for detecting 8-hydroxyguanine DNA glycosidase of the invention comprises:
a first electrode for generating an electrical signal for detecting 8-hydroxyguanine DNA glycosidase; the surface of the first electrode is modified with CP-DNA and TD-DNA combined through base complementary pairing.
Preferably, it further comprises:
a second electrode which constitutes a battery with the first electrode;
a third electrode having a constant potential to control a potential of the first electrode.
Preferably, before complementary pairing and further combination, the surface of the first electrode is sequentially subjected to cleaning pretreatment including goby solution soaking, distilled water washing, polishing, ultrasonic cleaning and electrochemical cleaning.
Wherein the goby solution is prepared from 98 percent of H2SO4And a concentration of 30% H2O2Prepared according to the ratio of 3: 1.
A method for detecting 8-hydroxyguanine DNA glycosidase by a sensor, which comprises the following steps:
modifying CP-DNA to the surface of the first electrode;
complementary pairing of the TD-DNA and the CP-DNA, the tail end of which is modified with a ferrocene group, and further combining the TD-DNA and the CP-DNA to the surface of the first electrode to obtain a first electrode containing a ferrocene electric signal;
immersing a first electrode containing a ferrocene electric signal into buffer solution containing hOGG1 with different concentrations, and carrying out 8-hydroxyguanine cutting and hybridization chain reaction to obtain the first electrode containing a hexa-ammonium ruthenium electric signal;
and reading the differential pulse voltammetric signal on the soaked first electrode, and calculating the relationship between the ferrocene electrical signal value variation, the hexaammonium ruthenium electrical signal value variation and the concentration of hOGG 1.
Preferably, before the CP-DNA is modified on the surface of the first electrode, the method further comprises the following steps of washing and pretreating the first electrode:
soaking the first electrode in the goby solution for 5 minutes, and then washing the first electrode with distilled water;
polishing the first electrode after being washed by 3000 meshes of sand paper and 5000 meshes of sand paper and polishing alumina powder with the particle size of 1.0 mu m, 0.3 mu m and 0.05 mu m to be smooth;
ultrasonically cleaning the polished first electrode in ethanol and distilled water for 5 minutes respectively;
the first electrode after ultrasonic cleaning was exposed to 50% HNO3Soaking in the solution for 30 min at 0.5M H2SO4Electrochemical cleaning is carried out in the solution.
Wherein the goby solution is prepared from 98 percent of H2SO4And a concentration of 30% H2O2Prepared according to the ratio of 3: 1.
Preferably, the CP-DNA is modified to the surface of the first electrode, comprising the steps of:
reacting the first electrode with a solution containing 0.5. mu.M CP-DNA at 25 ℃ for 16 hours to complete the modification;
the modified first electrode was sequentially subjected to soaking in 1mM mercaptoethanol for one hour, rinsing in distilled water, and drying in a nitrogen stream.
Preferably, the TD-DNA modified with ferrocene group at the end and the CP-DNA are complementarily paired and then combined to the surface of the first electrode, and the method comprises the following steps:
the CP-DNA modified first electrode was reacted with a 1. mu.M solution of TD-DNA end-modified with a ferrocenyl group at 37 ℃ for 1 hour.
Preferably, the first electrode containing ferrocene electrical signal is immersed in buffer containing hOGG1 with different concentration to perform 8-hydroxyguanine cleavage and hybridization chain reaction, comprising the steps of:
soaking a first electrode containing ferrocene electric signals in hOGG1 solutions containing different concentrations at 37 ℃ for reaction for 1 hour;
the first electrode was soaked in a solution containing 0.5. mu.M Linker DNA, 5. mu. M H1 and 5. mu. M H2 at 25 ℃ for 2 hours.
Preferably, in the range of 0.002U/mL to 10U/mL, the relationship between the ferrocene electrical signal value variation, the ruthenium hexammoniate electrical signal value variation and the concentration of hOGG1 satisfies the regression equation: y is 0.551x + 0.52;
wherein y is the logarithm of the ratio of the change amount of the hexa-ammonium ruthenium electrical signal value to the change amount of the ferrocene electrical signal value, and x is the logarithm of the concentration of hOGG 1.
Preferably, the method further comprises the following steps:
a second electrode which constitutes a battery with the first electrode;
a third electrode having a constant potential to control a potential of the first electrode. The invention at least comprises the following beneficial effects:
1) the sensor for detecting 8-hydroxyguanine DNA glycosidase is provided with the electrodes, the surfaces of the electrodes are complementarily paired, and then CP-DNA and TD-DNA with ferrocene group modified at the tail end are combined, so that the detection limit of the concentration of a substance to be detected hOGG1 is greatly reduced to 0.0008U/mL, and the sensor has remarkable specificity and higher stability;
2) the detection method of the sensor for detecting 8-hydroxyguanine DNA glycosidase provided by the invention comprises the following steps: complementary pairing of the TD-DNA and the CP-DNA, the tail end of which is modified with a ferrocene group, and further combining the TD-DNA and the CP-DNA to the surface of a first electrode to obtain the first electrode containing a ferrocene electric signal; immersing a first electrode containing a ferrocene electric signal into a solution containing hOGG1 with different concentrations, and carrying out 8-hydroxyguanine cutting and hybridization chain reaction to obtain a first electrode containing a hexa-ammonium ruthenium electric signal; reading the differential pulse voltammetric signal on the soaked first electrode, and calculating the relationship between the ferrocene electrical signal value variation, the hexaammonium ruthenium electrical signal value variation and the concentration of hOGG 1; on one hand, the method adopts an isothermal amplification method of a hybrid chain reaction to realize signal amplification, further improves the detection sensitivity and greatly reduces the cost; on the other hand, the adopted first electrode which is complementary and paired on the surface and further combined with CP-DNA and TD-DNA modified with ferrocene group at the tail end belongs to a ratio type electrochemical biosensor, and the detection limit of the concentration of the substance to be detected hOGG1 is greatly reduced to 0.0008U/mL; moreover, the biosensor is combined with a hybridization chain reaction, so that the biosensor has remarkable specificity and higher stability; the method is also suitable for detecting the activity of hOGG1 in complex biological samples such as serum and the like, and is a method for rapidly detecting the activity of hOGG1 with high selectivity and sensitivity.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram of the detection of a sensor for detecting 8-hydroxyguanine DNA glycosidase according to the present invention;
FIG. 2 is a graph of the AC impedance of the first electrode of the present invention at various stages of modification;
FIGS. 3(a) -3(b) are graphs of differential pulse voltammetric signals of ferrocene electrical signal and hexaammoniumtrutheniumhydroxide electrical signal measured before and after adding buffer containing hOGG1 to the first electrode of the present invention;
FIGS. 4(a) -4(b) are differential pulse voltammograms of an electrical signal of ferrocene and an electrical signal of ruthenium hexaammonium measured by adding buffers containing different concentrations of hOGG1 to a first electrode according to the present invention;
FIG. 5(a) is a graph of ferrocene electrical signal versus concentration of hOGG1 in accordance with the present invention;
FIG. 5(b) is the electrical signal value of ruthenium hexammoniate corresponding to the concentration of hOGG1 in accordance with the present invention;
FIG. 5(c) is the ratio of the electrical signal value of ruthenium hexammoniate to the electrical signal value of ferrocene corresponding to the concentration of hOGG1 in accordance with the present invention;
FIG. 6 shows the relative excess (10U/mL) of 1U/mL hOGG1 to the other interfering enzymes: the detection effects of restriction enzyme (EcoRI), DNA polymerase (Bst2.0DNApolymerase), and Transglutaminase (TG);
FIG. 7 is a flowchart of the detection by the sensor for detecting 8-hydroxyguanine DNA glycosidase according to the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
< embodiment 1>
The invention provides a sensor for detecting 8-hydroxyguanine DNA glycosidase, which comprises a first electrode for generating an electric signal for detecting 8-hydroxyguanine DNA glycosidase, and a CP-DNA and a TD-DNA combined through base complementary pairing modified on the surface of the first electrode, as shown in figure 1.
Specifically, in this embodiment, a ratio-type electrochemical method is adopted, CP-DNA is modified to the surface of the first electrode through a gold thiol bond, and TD-DNA modified with a ferrocenyl group at the end is complementarily paired with CP-DNA to be bound to the surface of the first electrode. At this time, the first electrode surface gave a significant ferrocene electrical signal due to the presence of a large number of electrochemically active ferrocene moieties. The first electrode is immersed in a buffer solution containing a sample to be tested hOGG1, and hOGG1 in the buffer solution can specifically recognize an 8-oxoG site on TD-DNA and realize cutting. The cut TD-DNA falls off from the surface of the first electrode, the ferrocene group is far away from the surface of the first electrode to cause the obvious weakening of ferrocene electric signals, and the CP-DNA modified on the surface of the first electrode exposes a viscous terminal to cause the hybridization chain reaction on the surface of the first electrode. The hybridization chain reaction product on the surface of the first electrode adsorbs electroactive material ruthenium hexaammonium, and obvious ruthenium hexaammonium electrical signals can be generated. Further, the electrochemical workstation CHI660D reads out the differential pulse voltammetric signal on the first electrode, and the change of the ferrocene electrical signal value and the change of the hexaammonium ruthenium electrical signal value after the sample to be detected is added can be calculated. Through calculation and analysis, the logarithm of the ratio of the increase of the hexammoniate ruthenium electrical signal value to the decrease of the ferrocene electrical signal value and the logarithm of the concentration of hOGG1 have a linear relation in a certain range.
Therefore, the first electrode with the surface complementarily paired with the CP-DNA and the TD-DNA with the tail end modified with the ferrocene group belongs to a ratio type electrochemical biosensor, and acquires electric signals comprising ferrocene and hexa-ammonium ruthenium in the process of detecting the activity of 8-hydroxyguanine DNA glycosidase; and then, the amplification of an electric signal is realized through a hybridization chain reaction generated on the surface of the first electrode in the detection, so that the detection limit of the concentration of the substance to be detected hOGG1 is greatly reduced to 0.0008U/mL, the sensitivity is high, the speed is high, the cost is low, the specificity is obvious, the stability is higher, the method is also suitable for detecting the activity of hOGG1 in complex biological samples such as serum, and an important basis is provided for the relation between a DNA repair mechanism and the tumor.
It should be added that the first electrode can be an electrode of different materials and different sizes, and it is only required to insert the buffer solution containing the sample to be tested hOGG1 and generate two electric signals of ferrocene and ruthenium hexaammonium after the first electrode is combined with CP-DNA and TD-DNA modified with ferrocene group at the end after the first electrode meets the complementary pairing. In this embodiment, a gold electrode having a diameter of 2mm is preferable.
Preferably, in the above embodiment, the sensor for detecting 8-hydroxyguanine DNA glycosidase further includes a second electrode and a third electrode. The embodiment provides a three-electrode system comprising a first electrode, a second electrode and a third electrode, wherein the first electrode is a working electrode, the second electrode is a counter electrode, and the second electrode and the first electrode form a battery to form a passage for testing current; the third electrode is a reference electrode having a constant potential, and the first electrode and the third electrode form another path for detecting the potential of the first electrode to control the potential of the first electrode. More preferably, the three electrodes are made of gold, platinum, and silver-silver chloride in this order.
Preferably, before complementary pairing and combination, the first electrode surface is sequentially subjected to cleaning pretreatment including goby solution soaking, distilled water washing, polishing, ultrasonic cleaning and electrochemical cleaning, wherein the goby solution is formed by 98% H2SO4And a concentration of 30% H2O2Prepared according to the ratio of 3: 1. The embodiment respectively carries out physical cleaning and chemical cleaning on the first electrode, prepares for the subsequent modification of CP-DNA to the surface of the first electrode through a gold sulfhydryl bond, and the complementary pairing of TD-DNA and CP-DNA of which the tail ends are modified with ferrocenyl groups so as to be combined to the surface of the first electrode, and improves the specificity recognition and cutting of hOGG1, the obvious weakening of ferrocene electric signals after cutting and the detection accuracy of the obvious increase of hexa-ammonium ruthenium electric signals after hybridization chain reaction.
< embodiment 2>
In addition to the sensor for detecting 8-hydroxyguanine DNA glycosidase provided in embodiment 1, the present embodiment provides a method for detecting 8-hydroxyguanine DNA glycosidase by the sensor, as shown in fig. 7, including the steps of:
s1, modifying the CP-DNA to the surface of the first electrode;
s2, carrying out complementary pairing on the TD-DNA and the CP-DNA of which the tail ends are modified with ferrocene groups so as to be combined to the surface of the first electrode, and obtaining the first electrode containing ferrocene electric signals;
s3, immersing the first electrode containing the ferrocene electric signal into buffer solution containing hOGG1 with different concentrations, and performing 8-hydroxyguanine cutting and hybridization chain reaction to obtain the first electrode containing the hexa-ammonium ruthenium electric signal;
and S4, reading the differential pulse voltammetric signal on the soaked first electrode, and calculating the relationship between the change of the ferrocene electrical signal value, the change of the hexaammonium ruthenium electrical signal value and the concentration of hOGG 1.
In the above embodiment, the first electrode having the surface complementarily paired to bind CP-DNA and TD-DNA having a ferrocenyl moiety modified at the terminal thereof is manufactured through steps S1 and S2, and belongs to a ratiometric electrochemical biosensor, in which electrical signals including ferrocene and ruthenium hexammoniate are collected during the detection of 8-hydroxyguanine DNA glycosidase activity; and then, the amplification of an electric signal is realized through a hybridization chain reaction generated on the surface of the first electrode in the detection, so that the detection limit of the concentration of the substance to be detected hOGG1 is greatly reduced to 0.0008U/mL, the sensitivity is high, the speed is high, the cost is low, the specificity is obvious, the stability is higher, the method is also suitable for detecting the activity of hOGG1 in complex biological samples such as serum, and an important basis is provided for the relation between a DNA repair mechanism and the tumor.
Preferably, the method further comprises, before step S1, before the CP-DNA is modified on the surface of the first electrode:
and S0, performing cleaning pretreatment on the first electrode.
Step S0 further includes:
soaking the first electrode in the goby solution for 5 minutes, and then washing the first electrode with distilled water;
polishing the first electrode after being washed by 3000 meshes of sand paper and 5000 meshes of sand paper and polishing alumina powder with the particle size of 1.0 mu m, 0.3 mu m and 0.05 mu m to be smooth;
ultrasonically cleaning the polished first electrode in ethanol and distilled water for 5 minutes respectively;
the first electrode after ultrasonic cleaning was exposed to 50% HNO3Soaking in the solution for 30 min at 0.5M H2SO4Electrochemical cleaning is carried out in the solution.
Wherein the goby solution is composed of 98% H2SO4And a concentration of 30% H2O2Prepared according to the ratio of 3: 1.
The embodiment respectively carries out physical cleaning and chemical cleaning on the first electrode, prepares for the subsequent modification of CP-DNA to the surface of the first electrode through a gold sulfhydryl bond, and the complementary pairing of TD-DNA and CP-DNA of which the tail ends are modified with ferrocenyl groups so as to be combined to the surface of the first electrode, and improves the specificity recognition and cutting of hOGG1, the obvious weakening of ferrocene electric signals after cutting and the detection accuracy of the obvious increase of hexa-ammonium ruthenium electric signals after hybridization chain reaction.
Preferably, the step S1 of modifying the CP-DNA on the surface of the first electrode includes the steps of:
the first electrode was reacted with a solution containing 0.5. mu.M CP-DNA at 25 ℃ for 16 hours to complete the modification;
the modified first electrode was sequentially subjected to soaking in 1mM mercaptoethanol for one hour, rinsing in distilled water, and drying in a nitrogen stream.
Preferably, in step S2, the complementary pairing between TD-DNA modified with a ferrocene group at its end and CP-DNA is further bonded to the surface of the first electrode, and the method includes the steps of:
the CP-DNA modified first electrode was reacted with a 1. mu.M solution of TD-DNA end-modified with a ferrocenyl group at 37 ℃ for 1 hour.
Preferably, in step S3, the first electrode containing ferrocene electric signal is immersed in buffer containing different concentrations of hgg 1 to perform 8-hydroxyguanine cleavage and hybridization chain reaction, including the steps of:
soaking a first electrode containing ferrocene electric signals in hOGG1 solutions containing different concentrations at 37 ℃ for reaction for 1 hour;
the first electrode was soaked in a solution containing 0.5. mu.M Linker DNA, 5. mu. M H1 and 5. mu. M H2 at 25 ℃ for 2 hours.
Further, in step S3, where the hybridization chain reaction is generated at the first electrode, to prove this, the buffer containing hOGG1 was set to 5mM [ Fe (CN) ] containing 1mM KCl6]3-/4-The solution is prepared by mixing a solvent and a solvent,FIG. 2 shows AC impedance plots of the first electrode at various stages of modification; for characterizing different modification stages of the electrode surface [ Fe (CN)6]3-/4-While preliminarily verifying the feasibility of the invention.
Specifically, in fig. 2, a curve a is the impedance of a bare first electrode, a curve b is the impedance after CP-DNA modification, a curve c is the impedance after CP-DNA/MCH modification, a curve d is the impedance after interaction with TD-DNA, a curve e is the impedance after interaction with hcogg 1, and a curve f is the impedance after interaction with Linker DNA, H1, H2. A typical impedance spectrum contains different linear and semi-circular portions corresponding to diffusion states and electron transfer processes. As shown in fig. 2, no distinct semicircular region was observed on the bare first electrode (curve a) due to the balance of steric hindrance and positive charge; a smaller semicircular area appears on the first electrode (curve b) for CP-DNA modification, which indicates that CP-DNA modification to the surface of the first electrode hinders charge transfer; after the first electrode was incubated with mercaptoethanol, the semicircular area was further enlarged (curve c); after the first electrode and the TD-DNA react, the semicircular area becomes larger further (curve d), which shows that the CP-DNA and the TD-DNA on the surface of the first electrode are subjected to complementary pairing, and the charge transfer on the surface of the first electrode is further hindered; the semicircular area becomes smaller after the first electrode is incubated with the sample containing hOGG1 (curve e), which indicates that hOGG1 can specifically recognize 8-oxoG on the cleaved TD-DNA, and the cleaved TD-DNA will be detached from the surface of the first electrode, resulting in a decrease in impedance. The semicircular area becomes significantly larger (curve f) after the first electrode is reacted with Linker DNA, H1, H2, which indicates that chain hybridization reaction occurs on the surface of the first electrode, and the product thereof significantly hinders the transfer of the surface charge of the first electrode.
As a preference of the above embodiment, the reading of the differential pulse voltammetric signal on the first electrode after soaking mentioned in step S4 can be read by the electrochemical workstation CHI660D, and in order to verify the feasibility of the method, fig. 3 shows an example of the differential pulse voltammetric graph measured before and after the first electrode is added with the hcogg 1, and fig. 3(a) is a graph of the differential pulse voltammetric signal of the ferrocene electrical signal measured before and after the first electrode is added with the buffer solution containing hcogg 1; FIG. 3(b) is theDifferential pulse voltammogram of the hexaammoniumtride electrical signal measured before and after one electrode addition to buffer containing hOGG 1. In this case, the buffer containing hOGG1 was 10mM PBS and 50. mu.M [ Ru (NH) ]3)6]3+10mM Tris-HCl solution.
Specifically, in FIG. 3(a), after 1U/mL of hOGG1 is added, the ferrocene electrical signal is significantly reduced, which indicates that hOGG1 exerts the enzyme digestion activity, specifically cleaves 8-oxoG on TD-DNA, and the cleaved TD-DNA is separated from the electrode surface, so that the ferrocene electrical signal is significantly reduced. In FIG. 3(b), when 1U/mL of hOGG1 was added, the ruthenium hexammoniate electrical signal increased significantly, which indicates that hOGG1 specifically cleaves 8-oxoG on TD-DNA, the cleaved TD-DNA will detach from the first electrode surface, and the CP-DNA on the first electrode surface will expose the sticky end to allow chain hybridization reaction on the first electrode surface. A large number of hybrid chain reaction products will adsorb the electroactive species ruthenium hexammoniate, producing a significant ruthenium hexammoniate electrical signal.
Because the hOGG1 can specifically recognize and cut 8-oxoG sites, and further initiate the chain hybridization reaction on the surface of the first electrode, the chain hybridization product on the surface of the first electrode realizes the amplification of the electric signal on the surface of the first electrode by adsorbing hexaammonium ruthenium, different hOGG1 concentrations can be evaluated by differential pulse voltammetry reading, and meanwhile, the detection sensitivity can be further improved by the ratio of the two electric signals. FIGS. 4(a) - (b) show the differential pulse voltammograms obtained for different concentrations of hOGG1, FIG. 4(a) is the differential pulse voltammogram of ferrocene electrical signal, FIG. 4(b) is the differential pulse voltammogram of hexa-ammoniumtruthenium electrical signal, and the concentrations of hOGG1 are 0, 0.01U/mL, 0.05U/mL, 0.25U/mL, 1U/mL, 5U/mL, and 10U/mL, respectively. As can be seen from fig. 4(a) - (b), the ferrocene electrical signal decreases with increasing concentration of hcogg 1, and the ruthenium hexaammonium electrical signal increases with increasing concentration of hcogg 1.
Further, in step S4, the relationship between the amount of change in the ferrocene electrical signal value, the amount of change in the hexaammoniumtuthenium electrical signal value, and the concentration of hOGG1 in the range of 0.002U/mL to 10U/mL satisfies the regression equation: y is 0.551x + 0.52; wherein y is the logarithm of the ratio of the variation of the hexammoniated ruthenium electrical signal value to the variation of the ferrocene electrical signal value, and x is the logarithm of the concentration of hOGG 1;
in order to further verify that the linear relationship is satisfied between the ferrocene electrical signal value variation, the ruthenium hexammoniate electrical signal value variation and the concentration of hOGG1, calibration graphs of electrical signal values corresponding to the concentration of hOGG1 are shown in FIGS. 5(a) - (c). Wherein, FIG. 5(a) is the ferrocene electrical signal value corresponding to the concentration of hOGG 1; FIG. 5(b) is the corresponding electrical signal value of ruthenium hexammoniate (Ru) 1 concentration; FIG. 5(c) is the ratio of the electrical signal value of ruthenium hexammoniate to the electrical signal value of ferrocene corresponding to the concentration of hOGG 1; error bars represent the relative standard deviation of three independent measurements; r is the standard deviation and n is the number of repetitions; r2＝0.991，n＝3。
Specifically, as shown in fig. 5(a), the amount of change in the ferrocene electrical signal value in the range of 0.01U/mL to 10U/mL showed a linear relationship with the logarithm of the level of hcogg 1 with the regression equation of-0.39 x +0.69(R ═ 0.39x +0.69 (R)20.977, n-3), where y is the amount of change in the ferrocene electrical signal value and x is the logarithm of the concentration of hcogg 1. Fig. 5(b), the variation of the electrical signal value of ruthenium hexammoniate in the range of 0.01U/mL to 10U/mL shows a linear relationship with the logarithm of the level of hgg 1, with the regression equation of y being 0.993x +2.14(R ═ g1 (R) (-)20.964, n-3), where y is the change in the electrical signal value for ruthenium hexammoniate, and x is the logarithm of the concentration of hOGG 1. As shown in FIG. 5(c), the logarithm of the ratio of the change amount of the ruthenium hexammoniate electrical signal value to the change amount of the ferrocene electrical signal value in the range of 0.002U/mL to 10U/mL shows a linear relationship with the logarithm of the level of hOGG1, and the regression equation is that y is 0.551x +0.52(R is20.991, n-3), where y is the logarithm of the ratio of the change in the electrical signal value of ruthenium hexammoniate to the change in the electrical signal value of ferrocene, and x is the logarithm of the concentration of hOGG 1. From the results, it is clear that the linear range is significantly increased and the detection limit is significantly reduced by the ratio of the two electrical signals.
Preferably, in the above embodiment, the sensor for detecting 8-hydroxyguanine DNA glycosidase further includes a second electrode and a third electrode. The embodiment provides a three-electrode system comprising a first electrode, a second electrode and a third electrode, wherein the first electrode is a working electrode, the second electrode is a counter electrode, and the second electrode and the first electrode form a battery to form a passage for testing current; the third electrode is a reference electrode having a constant potential, and the first electrode and the third electrode form another path for detecting the potential of the first electrode to control the potential of the first electrode. More preferably, the three electrodes are made of gold, platinum, and silver-silver chloride in this order.
It should be added that, in order to prove that the invention has remarkable specificity and high selectivity, fig. 6 shows that 1U/mlogg 1 is in excess (10U/mL) of other interfering enzymes: examples of the effects of detection by restriction enzyme (EcoRI), DNA polymerase (Bst2.0DNApolymerase), and Transglutaminase (TG).
Specifically, in FIG. 6, there is a significant difference in electrochemical signal between the assay of hOGG1 and the excess interfering enzyme, and this experimental result indicates that the method has significant specificity. Meanwhile, detection experiments in a buffer solution system and a serum system prove that the method can realize the detection of hOGG1 in complex samples such as serum and the like. Therefore, the experimental results show that hOGG1 specifically recognizes 8-oxoG, further confirming the high selectivity of the method.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.
Claims (9)
1. A method for detecting a sensor that detects 8-hydroxyguanine DNA glycosidase, the sensor comprising:
a first electrode for generating an electrical signal for detecting 8-hydroxyguanine DNA glycosidase; the surface of the first electrode is modified with CP-DNA and TD-DNA combined through base complementary pairing;
the method for detecting the sensor for detecting the 8-hydroxyguanine DNA glycosidase comprises the following steps:
modifying CP-DNA to the surface of the first electrode;
complementary pairing of the TD-DNA and the CP-DNA, the tail end of which is modified with a ferrocene group, and further combining the TD-DNA and the CP-DNA to the surface of the first electrode to obtain a first electrode containing a ferrocene electric signal;
immersing a first electrode containing a ferrocene electric signal into buffer solution containing hOGG1 with different concentrations, and carrying out 8-hydroxyguanine cutting and hybridization chain reaction to obtain the first electrode containing a hexa-ammonium ruthenium electric signal;
and reading the differential pulse voltammetric signal on the soaked first electrode, and calculating the relationship between the ferrocene electrical signal value variation, the hexaammonium ruthenium electrical signal value variation and the concentration of hOGG 1.
2. The method for detecting 8-hydroxyguanine DNA glycosidase according to claim 1, further comprising:
a second electrode which constitutes a battery with the first electrode;
a third electrode having a constant potential to control a potential of the first electrode.
3. The method for detecting the sensor of 8-hydroxyguanine DNA glycosidase according to claim 1, wherein before the complementary pairing and the combination, the surface of the first electrode is sequentially subjected to cleaning pretreatment comprising goby solution soaking, distilled water washing, polishing, ultrasonic cleaning and electrochemical cleaning;
wherein the goby solution is prepared from 98 percent of H2SO4And a concentration of 30% H2O2Prepared according to the ratio of 3: 1.
4. The method for detecting a sensor for detecting 8-hydroxyguanine DNA glycosidase according to claim 1, further comprising a washing pretreatment of the first electrode before the modification of CP-DNA to the surface of the first electrode, the washing pretreatment comprising the following steps:
soaking the first electrode in the goby solution for 5 minutes, and then washing the first electrode with distilled water;
polishing the first electrode after being washed by 3000 meshes of sand paper and 5000 meshes of sand paper and polishing alumina powder with the particle size of 1.0 mu m, 0.3 mu m and 0.05 mu m to be smooth;
ultrasonically cleaning the polished first electrode in ethanol and distilled water for 5 minutes respectively;
the first electrode after ultrasonic cleaning was exposed to 50% HNO3Soaking in the solution for 30 min at 0.5M H2SO4Carrying out electrochemical cleaning in the solution;
wherein the goby solution is prepared from 98 percent of H2SO4And a concentration of 30% H2O2Prepared according to the ratio of 3: 1.
5. The method for detecting 8-hydroxyguanine DNA glycosidase according to claim 1, wherein the CP-DNA is modified to the surface of the first electrode, comprising the steps of:
reacting the first electrode with a solution containing 0.5. mu.M CP-DNA at 25 ℃ for 16 hours to complete the modification;
the modified first electrode was sequentially subjected to soaking in 1mM mercaptoethanol for one hour, rinsing in distilled water, and drying in a nitrogen stream.
6. The method for detecting the detection of the sensor for detecting 8-hydroxyguanine DNA glycosidase according to claim 1, wherein the TD-DNA with end modified with ferrocene group is complementarily paired with the CP-DNA and then combined to the surface of the first electrode, comprising the steps of:
the CP-DNA modified first electrode was reacted with a 1. mu.M solution of TD-DNA end-modified with a ferrocenyl group at 37 ℃ for 1 hour.
7. The method for detecting 8-hydroxyguanine DNA glycosidase according to claim 1, wherein the first electrode containing ferrocene electric signal is immersed in buffers containing different concentrations of hOGG1 for 8-hydroxyguanine cleavage and hybridization chain reaction, comprising the steps of:
soaking a first electrode containing ferrocene electric signals in hOGG1 solutions containing different concentrations at 37 ℃ for reaction for 1 hour;
the first electrode was soaked in a solution containing 0.5. mu.M Linker DNA, 5. mu. M H1 and 5. mu. M H2 at 25 ℃ for 2 hours.
8. The method for detecting 8-hydroxyguanine DNA glycosidase according to claim 1, wherein the relationship between the amount of change of ferrocene electrical signal value, the amount of change of ruthenium hexaammonium electrical signal value and the concentration of hOGG1 in the range of 0.002U/mL to 10U/mL satisfies the regression equation: y is 0.551x + 0.52; wherein y is the logarithm of the ratio of the change amount of the hexa-ammonium ruthenium electrical signal value to the change amount of the ferrocene electrical signal value, and x is the logarithm of the concentration of hOGG 1.
9. The method for detecting 8-hydroxyguanine DNA glycosidase according to claim 1, further comprising:
a second electrode which constitutes a battery with the first electrode;
a third electrode having a constant potential to control a potential of the first electrode.
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