CN109142477B - Vibrio DNA electrochemical sensor and preparation method and application thereof - Google Patents
Vibrio DNA electrochemical sensor and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a vibrio DNA electrochemical sensor and a preparation method and application thereof. Stripping graphene oxide in an N, N-dimethylformamide solution containing amino ionic liquid to prepare ionic liquid functionalized graphene; then preparing hydrotalcite-like nano-sheets in the dispersion liquid to prepare an ionic liquid functionalized graphene-hydrotalcite-like nano-sheet compound, preparing a glassy carbon electrode modified by the compound by adopting a dripping method, and fixing a vibrio probe ssDNA on the surface of the modified electrode through a biological connecting agent to prepare the vibrio DNA sensor. The DNA sensor has good sensitivity and selectivity, lower detection limit and wider linear range. The preparation method comprises the following steps: preparing a modified electrode material; preparing an electrochemical vibrio parahaemolyticus DNA sensor; hybridizing the electrochemical vibrio parahemolyticus DNA sensor with the target DNA; and detecting electrochemical signals of the sensor.
Description
The technical field is as follows:
the invention belongs to the technical field of bioelectrochemical sensors, and particularly relates to a vibrio DNA electrochemical sensor, a preparation method thereof and specific DNA for high-sensitivity detection of pathogenic vibrio parahaemolyticus.
Background art:
marine vibrio is a rod-shaped or arc-shaped gram-negative bacterium with extremely strong activity, is widely distributed in brackish water, coastal areas, estuaries, marine water bodies, sediments and marine organisms, is a gram-negative bacterium distributed in the global estuarine environment, and can cause acute gastroenteritis of human beings when eating raw, uncooked or improperly processed seafood. The research shows that tlh genes are found in parahemolytic arc strains with the greatest harm of vibrio parahemolyticus, so that the tlh genes are regarded as a useful index for detecting the total vibrio parahemolyticus. In recent years, DNA electrochemical biosensors have attracted much attention due to their advantages such as low cost, high sensitivity, fast response speed, good selectivity, and miniaturization of instruments. To date, various types of DNA electrochemical biosensors have been used to improve the sensitivity and stability of DNA biosensors using a wide range of nanomaterials such as hydrotalcite-like compounds (LDHs), Carbon Nanotubes (CNTs), Graphene (GR), and the like, but DNA sensors for detecting vibrio still have much room for improvement in sensitivity and detection limit.
Graphene (GR) is an sp2In 2004, a two-dimensional material composed of hybridized carbon atoms and having a carbon atom thickness is successfully prepared by Geim for the first time, and has attracted extensive attention in various fields in recent years due to excellent mechanical properties and electrical properties. Chemical reduction is a low cost process and is therefore widely used for large scale industrial production of GR. However, it is not limited toDuring the production process, the GR sheets are irreversibly stacked due to strong pi-pi interaction, which not only impairs the dispersion properties of the GR, but also increases the contact resistance between the sheets, thereby severely limiting the electrochemical properties of the GR and its applications in various fields. The water-like slip (LDH) is a two-dimensional nano anionic clay, and the composition general formula can be expressed as [ M1-x 2+Mx 3+(OH)2]x+(An-)x/n·mH2O, having a hydrotalcite layered structure with sheets carrying positive charges and exchangeable anions present between the layers. The hydrotalcite-like nanosheets (LDHNS) exist in a single-layer or a plurality of-layer hydrotalcite-like nanosheets form, not only have various performances of the original LDH, but also have the characteristics of open inner surface, positive electricity layer with molecular thickness, large specific surface area, abundant active sites and the like, so that the application field of the LDH is greatly expanded. Therefore, researchers compound the LDHNS with positive charge and GR to prevent the two components from aggregating and improve the conductivity of the compound. Although the composite scheme can solve the problems of GR sheet stacking and LDHNS aggregation, the composite hybrid is easy to settle, the overall dispersibility is poor, and the conductivity of the electrode modified material is further improved. The amino functionalized ionic liquid is not only a high-conductivity green solvent with special solubility, but also has functional amino, so that the amino functionalized ionic liquid can be modified to the surface of a GO/GR-based composite material through ring opening reaction with a large number of epoxy rings on the surface of GO. Due to special solubility, a large amount of charges and high conductivity, the introduction of the ionic liquid can greatly improve the dispersibility, stability and conductivity of the GO/GR-based material.
According to the invention, GO is stripped by using amino functionalized ionic liquid, and LDHNS is synthesized in a formamide medium, so that an ionic liquid functionalized graphene-hydrotalcite-like nanosheet (IL-GR-LDHNS) compound is prepared, and a highly sensitive vibrio DNA hybridization detection method is established on the basis of a glassy carbon electrode modified by the IL-GR-LDHNS compound film. The IL-GR-LDHNS composite membrane not only can effectively fix DNA, but also has large specific surface area, abundant active sites, good biocompatibility, conductivity and dispersibility, and effectively promotes electron transfer. Compared with other reported methods, the method has the advantages of lower detection limit on target DNA, wide linear range and good selectivity, and can realize high-sensitivity unmarked detection on the vibrio characteristic DNA.
The invention content is as follows:
aiming at the defects of the existing DNA sensing detection technology and the requirements of research and application in the field, one of the purposes of the invention is to provide a vibrio DNA electrochemical sensor, wherein a glassy carbon electrode is used as a substrate electrode of the DNA sensor, an ionic liquid functionalized graphene-hydrotalcite-like nanosheet composite membrane is used as an electrode modification material, and a probe ssDNA is fixed on the surface of the modification electrode in a covalent bonding mode of a biological connecting agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide; the ionic liquid functionalized graphene-hydrotalcite-like nanosheet composite membrane is prepared by firstly stripping graphene oxide in an N, N-dimethylformamide solution containing amino ionic liquid to prepare ionic liquid functionalized graphene; then preparing hydrotalcite-like nano-sheets in the dispersion liquid to prepare an ionic liquid functionalized graphene-hydrotalcite-like nano-sheet compound; the glassy carbon electrode is marked as GCE; the N, N-dimethylformamide is taken as DMF; the graphene oxide is marked as GO, and the graphene is marked as GR; the hydrotalcite-like nano-sheet is marked as LDHNS; the ionic liquid is 1-methyl-3-aminobutylimidazolium tetrafluoroborate, is marked as IL, and has the following structural formula:
the invention also aims to provide a preparation method of the vibrio DNA electrochemical sensor, which is characterized by comprising the following specific steps:
(1) preparation of IL-GR-LDHNS Complex
Adding a certain amount of GO into 100mL of formamide containing 200mg of IL, and performing ultrasonic dispersion for 2 hours to obtain an IL-GO dispersion liquid with GO concentration of 0.5-1.5 mg/mL; adding MgCl into the mixture according to a certain molar ratio2·6H2O and AlCl3·9H2O, enabling the total metal ion concentration to be 0.03mol/L, stirring for 1h to completely dissolve metal salt, slowly titrating by using a formamide solution of sodium hydroxide with the concentration of 0.5mol/L under the stirring condition until the pH value of a reaction liquid is about 9.0-10.0, stirring for reaction for 12h at room temperature, then aging for 12h at 30-70 ℃, centrifuging the reaction liquid for 10min at the rotation speed of 8000rpm, washing for 3 times by using ethanol and deionized water respectively, and naturally drying at room temperature to obtain an ionic liquid functionalized graphene-hydrotalcite-like nanosheet compound, namely IL-GR-LDHNS;
(2) preparation of IL-GR-LDHNS modified glassy carbon electrode
Using 0.05 μm A1 as glassy carbon electrode2O3After polishing, the polishing powder is washed clean by double distilled water, is subjected to ultrasonic treatment in an ultrasonic water bath for 5min, and is dried by high-purity nitrogen; ultrasonically dispersing the IL-GR-LDHNS compound obtained in the step (1) in deionized water to prepare a dispersion liquid with the concentration of 3mg/mL, dropwise coating 0.5-50 mu L of the dispersion liquid on the surface of a treated glassy carbon electrode, and naturally airing to obtain an IL-GR-LDHNS modified glassy carbon electrode which is marked as IL-GR-LDHNS/GCE;
(3) preparation of DNA sensor
Immersing IL-GR-LDHNS/GCE into a mixed solution of 0.4 mol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1mol/L N-hydroxysuccinimide for 30min to carboxylate the surface of the modified electrode; 1-100 μ L of 1 × 10-6And dripping a mol/L probe ssDNA solution on the surface of the modified electrode, incubating for 2h at 25-70 ℃, sequentially washing the electrode with PBS (phosphate buffer solution) with the pH of 7.4 and double distilled water to remove the non-immobilized probe ssDNA, and naturally drying at room temperature to obtain the DNA sensor.
Wherein in step (1) MgCl2·6H2O and AlCl3·9H2The molar ratio of O is 2: 1; in the aging process, IL and epoxy rings on GO molecules generate ring-opening reaction in an alkaline medium, so that covalent modification and partial reduction of GO by IL are realized; the LDHNS prepared in the formamide medium has the ultrathin characteristic, and the thickness is about 0.7-2 nm; the glassy carbon electrode polished in the step (2) is detected by adopting a three-electrode system in [ Fe (CN)6]3-/4-In the solution, the voltage is set to be-0.4-0.8V, and the glassy carbon electrode is subjected to charge-dischargeAnd (3) performing cyclic voltammetry scanning, wherein if the potential difference of the oxidation reduction peak is within 0-100 mV, the electrode surface is well treated, otherwise, the electrode surface is treated again until the electrode surface meets the requirement.
The invention also aims to provide a vibrio DNA electrochemical sensor for detecting vibrio. The method specifically comprises the following specific steps:
(1) hybridization of DNA sensor to target ssDNA
And (3) dripping complementary DNA with different concentrations onto the surface of the electrode fixed with the probe, incubating for 50min at 35 ℃ for hybridization, washing the electrode by using PBS (pH is 7.4) and double distilled water in sequence after hybridization, and removing non-hybridized target DNA, namely completing the hybridization of DNA molecules on the surface of the electrode.
(2) Electrochemical signal detection of sensors
Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) are adopted for detection, different modified glassy carbon electrodes are used as working electrodes, a platinum wire electrode is used as a counter electrode, an Ag/AgCl electrode is used as a reference electrode, [ Fe (CN) ]6]3-/4-As an indicator, the detection base solution is 0-10 mmol L-1[Fe(CN)6]3-/4-And 0 to 1mol L-1KCl PBS (pH is 7-9) buffer solution, an electrochemical workstation is used for testing cyclic voltammetry curves and differential pulse voltammetry curves of different modified electrodes, the scanning potential is-1V, and the scanning speed is 1-200 mV/s. The peak profile change was observed and the reduction peak current value was recorded.
Compared with the prior art, the main advantages are that: the vibrio DNA electrochemical sensor fully exerts the synergistic effect of IL, GR and LDHNS, improves the specific surface area, conductivity and dispersity of the composite membrane, increases active sites, and greatly enhances the fixing capacity of a modified interface on the ssDNA of the probe, thereby integrally improving the performance of the DNA electrochemical sensor for detecting vibrio characteristic DNA, and having important theoretical significance and potential application value for high-sensitivity label-free detection of pathogenic vibrio parahaemolyticus. The sensor has high sensitivity and good selectivity, and particularly has a lower detection limit and a wider linear range; the preparation method is simple and the detection speed is high.
Description of the drawings:
FIG. 1 is the scanning electron micrographs of GO-LDHNS (a) obtained in step (1) of comparative example 4 and IL-GR-LDHNS (b) obtained in step (1) of example 1.
FIG. 2 shows the ratios of GCE, LDHNS/GCE, GO-LDHNS/GCE and IL-GR-LDHNS/GCE at 5mmol L for comparative example 1, comparative example 2, comparative example 3, comparative example 4 and example 1, respectively-1 [Fe(CN)6]3-/4-And 0.1mol L- 1CV results in PBS (pH 7.4) buffer of KCl.
FIG. 3 shows the ratios of GCE, LDHNS/GCE, GO-LDHNS/GCE and IL-GR-LDHNS/GCE at 5mmol L for comparative example 1, comparative example 2, comparative example 3, comparative example 4 and example 1, respectively-1 [Fe(CN)6]3-/4-And 0.1mol L- 1Electrochemical impedance results in KCl in PBS (pH 7.4) buffer.
FIG. 4 shows IL-GR-LDHNS/GCE (a) corresponding to example 1, after immobilization of ssDNA as a probe (b) and after hybridization with a target DNA (c) [ Fe (CN)6]3-/4-And CV in KCl in PBS.
FIG. 5 shows the DPV results of IL-GR-LDHNS/GCE (a), to which the probe ssDNA was immobilized, after hybridization with non-complementary DNA (b), three-base mismatched DNA (c), single-base mismatched DNA (d), and complete complementary DNA (e) according to example 1.
FIG. 6 shows the DPV response results (a-k) of different concentrations of target ssDNA hybridized to the corresponding DNA sensor of example 1 (0, 1.0X 10)-16，1.0×10-15，1.0×10-14，1.0×10-13，1.0×10-12， 1.0×10-11，1.0×10-10，1.0×10-9，1.0×10-8，1.0×10-7mol L-1The inset is Ipaand-lgC.
The specific implementation mode is as follows:
for a further understanding of the invention, reference will now be made to the following examples and drawings, which are not intended to limit the invention in any way.
Example 1:
(1) preparation of IL-GR-LDHNS Complex
Will be fixedAdding GO with the amount into 100mL of formamide containing 200mg of IL, and performing ultrasonic dispersion for 2 hours to obtain an IL-GO dispersion liquid with GO concentration of 1.0 mg/mL; according to the following steps: 1 molar ratio to which MgCl was added2·6H2O and AlCl3·9H2O, enabling the total metal ion concentration to be 0.03mol/L, stirring for 1h to completely dissolve metal salt, slowly titrating by using a formamide solution of sodium hydroxide with the concentration of 0.5mol/L under the stirring condition until the pH value of a reaction liquid is about 9.0-10.0, stirring for reaction for 12h at room temperature, then aging for 12h at 60 ℃, centrifuging the reaction liquid for 10min at the rotation speed of 8000rpm, washing for 3 times by using ethanol and deionized water respectively, and naturally drying at room temperature to obtain an ionic liquid functionalized graphene-hydrotalcite-like nanosheet compound, namely IL-GR-LDHNS;
(2) preparation of IL-GR-LDHNS modified glassy carbon electrode
Using 0.05 μm A1 as glassy carbon electrode2O3After polishing, the polishing powder is washed clean by double distilled water, is subjected to ultrasonic treatment in an ultrasonic water bath for 5min, and is dried by high-purity nitrogen; ultrasonically dispersing the IL-GR-LDHNS compound obtained in the step (1) in deionized water to prepare a dispersion liquid with the concentration of 3mg/mL, dropwise coating 12 mu L of the dispersion liquid on the surface of a treated glassy carbon electrode, and naturally airing to obtain an IL-GR-LDHNS modified glassy carbon electrode which is marked as IL-GR-LDHNS/GCE;
(3) preparation of DNA sensor
Immersing IL-GR-LDHNS/GCE into a mixed solution of 0.4 mol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1mol/L N-hydroxysuccinimide for 30min to carboxylate the surface of the modified electrode; taking 20 μ L of 1 × 10-6And dripping a mol/L probe ssDNA solution on the surface of the modified electrode, incubating for 2h at 25 ℃, sequentially washing the electrode with PBS (phosphate buffer solution) with the pH of 7.4 and double distilled water, removing the non-immobilized probe ssDNA, and naturally drying at room temperature to obtain the DNA sensor.
Example 2:
(1) preparation of IL-GR-LDHNS Complex
Adding a certain amount of GO into 100mL of formamide containing 200mg of IL, and performing ultrasonic dispersion for 2 hours to obtain an IL-GO dispersion liquid with GO concentration of 0.5 mg/mL; according to the following steps: 1 molar ratio to which MgCl was added2·6H2O and AlCl3·9H2O, enabling the total metal ion concentration to be 0.03mol/L, stirring for 1h to completely dissolve metal salt, slowly titrating by using a formamide solution of sodium hydroxide with the concentration of 0.5mol/L under the stirring condition until the pH value of a reaction liquid is about 9.0-10.0, stirring for reaction for 12h at room temperature, then aging for 12h at 70 ℃, centrifuging the reaction liquid for 10min at the rotation speed of 8000rpm, washing for 3 times by using ethanol and deionized water respectively, and naturally drying at room temperature to obtain an ionic liquid functionalized graphene-hydrotalcite-like nanosheet compound, namely IL-GR-LDHNS;
(2) preparation of IL-GR-LDHNS modified glassy carbon electrode
Using 0.05 μm A1 as glassy carbon electrode2O3After polishing, the polishing powder is washed clean by double distilled water, is subjected to ultrasonic treatment in an ultrasonic water bath for 5min, and is dried by high-purity nitrogen; ultrasonically dispersing the IL-GR-LDHNS compound obtained in the step (1) in deionized water to prepare a dispersion liquid with the concentration of 3mg/mL, dropwise coating 18 mu L of the dispersion liquid on the surface of a treated glassy carbon electrode, and naturally airing to obtain an IL-GR-LDHNS modified glassy carbon electrode which is marked as IL-GR-LDHNS/GCE;
(3) preparation of DNA sensor
Immersing IL-GR-LDHNS/GCE into a mixed solution of 0.4 mol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1mol/L N-hydroxysuccinimide for 30min to carboxylate the surface of the modified electrode; taking 40 μ L of 1 × 10-6And dripping a mol/L probe ssDNA solution on the surface of the modified electrode, incubating for 2h at 35 ℃, sequentially washing the electrode with PBS (phosphate buffer solution) with the pH of 7.4 and double distilled water, removing the non-immobilized probe ssDNA, and naturally drying at room temperature to obtain the DNA sensor.
Example 3:
(1) preparation of IL-GR-LDHNS Complex
Adding a certain amount of GO into 100mL of formamide containing 200mg of IL, and performing ultrasonic dispersion for 2 hours to obtain an IL-GO dispersion liquid with GO concentration of 1.5 mg/mL; according to the following steps: 1 molar ratio to which MgCl was added2·6H2O and AlCl3·9H2O, the total metal ion concentration is 0.03mol/L, the metal salt is completely dissolved by stirring for 1h, and the concentration is 0.5 under the stirring conditionSlowly titrating a mol/L formamide solution of sodium hydroxide until the pH of a reaction solution is about 9.0-10.0, stirring at room temperature for 12h, then aging at 60 ℃ for 12h, centrifuging the reaction solution at 8000rpm for 10min, washing with ethanol and deionized water for 3 times respectively, and naturally drying at room temperature to obtain an ionic liquid functionalized graphene-hydrotalcite-like nanosheet compound, which is marked as IL-GR-LDHNS;
(2) preparation of IL-GR-LDHNS modified glassy carbon electrode
Prepared according to the method and conditions of step (1) in example 1;
(3) preparation of DNA sensor
Immersing IL-GR-LDHNS/GCE into a mixed solution of 0.4 mol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1mol/L N-hydroxysuccinimide for 30min to carboxylate the surface of the modified electrode; taking 60 μ L of 1 × 10-6And dripping a mol/L probe ssDNA solution on the surface of the modified electrode, incubating for 2h at 45 ℃, sequentially washing the electrode with PBS (phosphate buffer solution) with the pH of 7.4 and double distilled water, removing the non-immobilized probe ssDNA, and naturally drying at room temperature to obtain the DNA sensor.
Example 4:
(1) preparation of IL-GR-LDHNS Complex
Prepared according to the method and conditions of step (1) in example 1;
(2) preparation of IL-GR-LDHNS modified glassy carbon electrode
Prepared according to the method and conditions of step (2) in example 1;
(3) preparation of DNA sensor
Immersing IL-GR-LDHNS/GCE into a mixed solution of 0.4 mol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1mol/L N-hydroxysuccinimide for 30min to carboxylate the surface of the modified electrode; taking 20 μ L of 1 × 10-6And dripping a mol/L probe ssDNA solution on the surface of the modified electrode, incubating for 2h at 55 ℃, sequentially washing the electrode with PBS (phosphate buffer solution) with the pH of 7.4 and double distilled water, removing the non-immobilized probe ssDNA, and naturally drying at room temperature to obtain the DNA sensor.
Comparative example 1:
the probe ssDNA was directly immobilized on the surface of the naked GCE according to the method and conditions of step (3) in example 1.
Comparative example 2:
(1) preparation of LDHNS slurry
According to the following steps: 1 molar of MgCl2·6H2O and AlCl3·9H2Adding O into 100mL of formamide to enable the total metal ion concentration to be 0.03mol/L, stirring for 1h to enable metal salts to be completely dissolved, slowly titrating formamide solution of sodium hydroxide with the concentration of 0.5mol/L under the stirring condition until the pH of the reaction solution is about 9.0-10.0, stirring at room temperature for 12h, then aging at 60 ℃ for 12h, centrifuging the reaction solution at 8000rpm for 10min, washing with ethanol and deionized water for 3 times respectively, and marking the slurry as LDHNS;
(2) preparation of LDHNS modified GCE
Preparing LDHNS dispersion liquid with the concentration of 3mg/mL according to the method and the conditions in the step (2) in the embodiment 1, taking 12 mu L of the dispersion liquid to be dripped on the surface of the treated GCE, and naturally airing at room temperature to obtain LDHNS/GCE;
(3) preparation of DNA sensor
The probe ssDNA was immobilized on the LDHNS/GCE surface according to the method and conditions of step (3) in example 1.
Comparative example 3:
(1) preparation of GO modified GCE electrode
Preparing GO dispersion liquid with the concentration of 3mg/mL according to the method and conditions in the step (2) in the embodiment 1, dropwise coating 12 mu L of the dispersion liquid on the surface of the processed GCE, and naturally airing at room temperature to obtain GO/GCE;
(2) preparation of DNA sensor
The probe ssDNA was immobilized on a bare GO/GCE surface according to the method and conditions of step (3) in example 1.
Comparative example 4:
(1) preparation of GO-LDHNS composite
Adding a certain amount of GO into 100mL of formamide, and performing ultrasonic dispersion for 2 hours to obtain GO dispersion liquid with the concentration of 1.0 mg/mL; according to the following steps: 1 molar ratio to which MgCl was added2·6H2O and AlCl3·9H2O, to make the total metal ion concentration 003mol/L, stirring for 1h to completely dissolve metal salt, slowly titrating by using formamide solution of sodium hydroxide with the concentration of 0.5mol/L under the stirring condition until the pH of the reaction liquid is about 9.0-10.0, stirring at room temperature for reaction for 12h, then aging at 60 ℃ for 12h, centrifuging the reaction liquid at the rotation speed of 8000rpm for 10min, washing with ethanol and deionized water for 3 times respectively, and naturally drying at room temperature to obtain the graphene oxide-hydrotalcite nanosheet compound, which is marked as GO-LDHNS;
(2) preparation of GO-LDHNS modified GCE electrode
Preparing GO-LDHNS dispersion liquid with the concentration of 3mg/mL according to the method and conditions in the step (2) in the embodiment 1, dropwise coating 12 mu L of the dispersion liquid on the surface of the processed GCE, and naturally airing at room temperature to obtain GO-LDHNS/GCE;
(3) preparation of DNA sensor
According to the method and conditions of step (3) in example 1, probe ssDNA is immobilized on the GO-LDHNS/GCE surface.
FIG. 1 is the scanning electron micrographs of GO-LDHNS (a) obtained in step (1) of comparative example 4 and IL-GR-LDHNS (b) obtained in step (1) of example 1. From (a), the LDHNS grows on the GO substrate, presents a sheet structure and has a certain aggregation phenomenon; when IL exists, the sheet materials in the IL-GO-LDHNS compound are connected together through the IL to present a continuous state, and the aggregation phenomenon is basically eliminated, thereby being beneficial to the electron transfer and mass transfer process of the modified electrode.
FIG. 2 shows the ratios of GCE, LDHNS/GCE, GO-LDHNS/GCE and IL-GR-LDHNS/GCE at 5mmol L for comparative example 1, comparative example 2, comparative example 3, comparative example 4 and example 1, respectively-1 [Fe(CN)6]3-/4-And 0.1mol L- 1CV results in PBS (pH 7.4) buffer of KCl. As can be seen from the figure, the GO and the LDHNS are not conductive, and the single LDHNS and GO have certain aggregation phenomenon to block the electron transfer of the redox probe, so that the CV curves of the LDHNS/GCE and the GO/GCE are not obviously improved compared with the redox response of the naked GCE. After LDHNS and GO are compounded, because the aggregation of the two flaky nano materials is mutually inhibited and more active sites are kept, the oxidation on GO-LDHNS/GCE is realizedThe reduction current is obviously increased. The IL-GR-LDHNS/GCE gives a obviously increased redox signal, which is mainly because the covalent modification of the IL not only improves the surface area and the active sites of the nanocomposite material, but also improves the conductivity and the dispersity of the material and improves the electrochemical catalytic activity of the material.
FIG. 3 shows the ratios of 5mmol L of GCE, LDHNS/GCE, GO-LDHNS/GCE and IL-GR-LDHNS/GCE for comparative example 1, comparative example 2, comparative example 3, comparative example 4 and example 1-1 [Fe(CN)6]3-/4-And 0.1mol L-1Electrochemical impedance results in KCl in PBS (pH 7.4) buffer. As can be seen from the figure, the GO and the LDHNS are not conductive, and the single LDHNS and GO have certain aggregation phenomenon, so that the mass transfer process of the electroactive substances is hindered. After the GO-LDHNS modifies the electrode, the two flaky nano-components mutually inhibit the self aggregation, and provide larger specific surface area and more active sites, so that the electrochemical impedance of the GO-LDHNS/GCE is further reduced. After GCE is modified by IL-GR-LDHNS, the electrochemical impedance of IL-GR-LDHNS/GCE is further reduced, the fastest electron transfer capability is shown, the ionic liquid functionalized nano composite improves the conductivity and the dispersity of a modified electrode, and the speed is increased [ Fe (CN) ]6]3-/4-Electron transfer of the probe.
Example 5:
and respectively dropwise adding complementary DNA with different concentrations to the surfaces of GCE, LDHNS/GCE, GO-LDHNS/GCE and IL-GR-LDHNS/GCE which are fixed with probe ssDNA and correspond to the comparative example 1, the comparative example 2, the comparative example 3, the comparative example 4 and the example 1, incubating for 50min at 35 ℃ for hybridization, washing the modified electrode by PBS with pH of 7.4 and double distilled water in sequence after hybridization, removing the target DNA which is hybridized, and completing the hybridization of DNA molecules on the surface of the electrode.
Using different modified GCE immobilized with probe ssDNA as working electrode, platinum wire electrode as counter electrode, Ag/AgCl electrode as reference electrode, [ Fe (CN)6]3-/4-As an indicator, the detection base solution is5mmol L-1 [Fe(CN)6]3-/4-And 0.1mol L-1KCl (pH 7.4) buffer solution in PBS is used for testing CV and DPV curves of different modified electrodes in an electrochemical workstation, the scanning potential is-0.4-0.8V, and the scanning speed is 100 mV/s. The peak profile change was observed and the reduction peak current value was recorded.
FIG. 4 shows IL-GR-LDHNS/GCE (a) corresponding to example 1, after immobilization of ssDNA as a probe (b) and after hybridization with a target DNA (c) [ Fe (CN)6]3-/4-And CV in KCl in PBS. It can be seen that the redox peak current is significantly reduced after immobilization of the probe ssDNA (b) relative to IL-GR-LDHNS/GCE (a), mainly due to the fact that the negatively charged phosphate backbone on the ssDNA surface repels the negatively charged [ Fe (CN) ]6]3-/4-The probe reached the electrode surface, indicating that the probe ssDNA was successfully immobilized on the modified electrode. After hybridization with the target DNA (c), the peak current is further reduced, which is attributable to the enhanced electrostatic repulsion between the negatively charged probe and the more negatively charged phosphate backbone after hybridization, and thus a lower electrochemical response. Obviously, the selective combination of the peak current change and the target DNA can be used as a sensing signal of the DNA sensor.
FIG. 5 shows the DPV results of IL-GR-LDHNS/GCE (a), to which the probe ssDNA was immobilized, after hybridization with non-complementary DNA (b), three-base mismatched DNA (c), single-base mismatched DNA (d), and complete complementary DNA (e) according to example 1. It can be seen that when the probe ssDNA immobilized IL-GR-LDHNS/GCE was hybridized with a single base mismatched DNA (d), the voltammetric response was enhanced compared to the result after hybridization with a fully complementary DNA (e). Similarly, the peak current signal is further enhanced after hybridization (c) of the probe ss DNA with the three-base mismatched DNA. The peak current signal becomes significantly greater when hybridized to fully non-complementary DNA (b), but the ssDNA-immobilized IL-GR-LDHNS/GCE (a) gives the maximum peak current. This indicates that the DNA sensor based on IL-GR-LDHNS/GCE has high selectivity, and can distinguish single-base, three-base mismatch and non-complementary DNA.
FIG. 6 shows the DPV response results (a-k) of different concentrations of target ssDNA hybridized to the corresponding DNA sensor of example 1 (0, 1.0X 10)-16，1.0×10-15，1.0×10-14，1.0×10-13，1.0×10-12， 1.0×10-11，1.0×10-10，1.0×10-9，1.0×10-8，1.0×10-7mol L-1The inset is Ipaand-lgC. It can be seen from the figure that the peak current signal becomes larger as the concentration of the target ssDNA decreases, because different concentrations of target ssDNA hybridize to the probe ssDNA to form dsDNA in different amounts, and the blocking effect on electron transfer is different. A smaller signal indicates more dsDNA is formed on the modified electrode surface. Inset is oxidation peak current value (I)pa) Linear dependence of the logarithm of the target DNA concentration (-lgC) on the negative value of the logarithm of the target DNA concentration (C), the target DNA concentration being 10-7～10-16mol L-1Between ranges, IpaThere is a good linear relationship with-lgC: i ispa(μA)＝2.76lg(C/M)–30.15，(R20.994) and a detection limit of 2.63 × 10-17mol L-1. The vibrio DNA sensor obtained by the invention has high sensitivity.
Table 1 shows the comparison of the analysis performance of the DNA sensor of the invention IL-GR-LDHNS/GCE vibrio with other DNA sensors
As can be seen from Table 1, compared with other electrochemical DNA sensors, the electrochemical DNA sensor based on the IL-GR-LDHNS/GCE provided by the invention has the advantages that the linear range is obviously increased, the detection limit is obviously reduced, and the IL-GR-LDHNSE nano composite membrane promotes electron transfer, increases the fixed quantity of probe DNA and reduces the detection limit.
Claims (2)
1. A vibrio DNA electrochemical sensor is characterized in that a glassy carbon electrode is used as a substrate electrode of the vibrio DNA electrochemical sensor, an ionic liquid functionalized graphene-hydrotalcite-like nanosheet composite membrane is used as an electrode modification material, and a probe ssDNA is fixed on the surface of the modification electrode in a covalent bonding mode through a biological connecting agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide; the ionic liquid functionalized graphene-hydrotalcite-like nanosheet composite membrane is prepared by firstly stripping graphene oxide in a formamide solution containing amino ionic liquid to prepare ionic liquid functionalized graphene, and then preparing hydrotalcite-like nanosheets in a dispersion solution containing the ionic liquid functionalized graphene to prepare an ionic liquid functionalized graphene-hydrotalcite-like nanosheet composite; the glassy carbon electrode is marked as GCE; the N, N-dimethylformamide is taken as DMF; the graphene oxide is marked as GO, and the graphene is marked as GR; the hydrotalcite-like nano-sheet is marked as LDHNS; the ionic liquid is 1-methyl-3-aminobutylimidazolium tetrafluoroborate, is marked as IL, and has the following structural formula:
the preparation method of the vibrio DNA electrochemical sensor comprises the following specific steps:
(1) preparation of IL-GR-LDHNS Complex
Adding a certain amount of GO into 100mL of formamide containing 200mg of IL, and performing ultrasonic dispersion for 2 hours to obtain an IL-GO dispersion liquid with GO concentration of 0.5-1.5 mg/mL; adding MgCl into the mixture according to a certain molar ratio2·6H2O and AlCl3·9H2O, enabling the total metal ion concentration to be 0.03mol/L, stirring for 1h to completely dissolve metal salt, slowly titrating by using a formamide solution of sodium hydroxide with the concentration of 0.5mol/L under the stirring condition until the pH of a reaction solution is 9.0-10.0, stirring for reaction for 12h at room temperature, then aging for 12h at 30-70 ℃, centrifuging the reaction solution for 10min at the rotation speed of 8000rpm, washing for 3 times by using ethanol and deionized water respectively, and naturally drying at room temperature to obtain an ionic liquid functionalized graphene-hydrotalcite-like nanosheet compound, namely IL-GR-LDHNS;
(2) preparation of IL-GR-LDHNS modified glassy carbon electrode
Using 0.05 μm A1 as glassy carbon electrode2O3Polishing with polishing powder, cleaning with double distilled water, ultrasonic treating in ultrasonic water bath for 5min, and blowing with high purity nitrogen gasDrying; ultrasonically dispersing the IL-GR-LDHNS compound obtained in the step (1) in deionized water to prepare a dispersion liquid with the concentration of 3mg/mL, dropwise coating 0.5-50 mu L of the dispersion liquid on the surface of a treated glassy carbon electrode, and naturally airing to obtain an IL-GR-LDHNS modified glassy carbon electrode which is marked as IL-GR-LDHNS/GCE;
(3) preparation of DNA sensor
Immersing IL-GR-LDHNS/GCE into a mixed solution of 0.4 mol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1mol/L N-hydroxysuccinimide for 30min to carboxylate the surface of the modified electrode; 1-100 μ L of 1 × 10- 6Dropwise coating a mol/L probe ssDNA solution on the surface of a modified electrode, incubating for 2h at 25-70 ℃, sequentially washing the electrode with PBS (phosphate buffer solution) with the pH of 7.4 and double distilled water to remove the non-immobilized probe ssDNA, and naturally drying at room temperature to obtain a DNA sensor;
the vibrio DNA electrochemical sensor is characterized in that MgCl is adopted in the step (1) of the preparation method2·6H2O and AlCl3·9H2The molar ratio of O is 2: 1; in the aging process, IL and epoxy rings on GO molecules generate ring-opening reaction in an alkaline medium, so that covalent modification and partial reduction of GO by IL are realized; the LDHNS prepared in the formamide medium has the ultrathin characteristic, and the thickness is 0.7-2 nm;
the glassy carbon electrode after polishing in the step (2) of the preparation method adopts a three-electrode system for detection, and is characterized in that [ Fe (CN)6]3-/4-Setting the voltage to be-0.4-0.8V in the solution, carrying out cyclic voltammetry scanning on the glassy carbon electrode, if the potential difference of an oxidation reduction peak is within 0-100 mV, indicating that the surface of the electrode is well treated, otherwise, carrying out retreatment until the surface meets the requirement.
2. The Vibrio DNA electrochemical sensor according to claim 1, which is used for detecting Vibrio.
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