CN111830109A - Preparation method of high-sensitivity photoelectric chemical sensor for detecting mucin - Google Patents
Preparation method of high-sensitivity photoelectric chemical sensor for detecting mucin Download PDFInfo
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- G—PHYSICS
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention discloses a preparation method of a high-sensitivity photoelectric chemical sensor for detecting mucin. By growing the composite nano material of titanium dioxide and sulfur indium zinc on the surface of the indium tin oxide conductive electrode modified with the gold nanoparticles, more hairpin DNA chains can be fixed, and the detection sensitivity is improved; then, the multi-branch hybrid chain which is connected with the aptamer and modified with manganese dioxide particles is loaded on the surface of the electrode, has good catalytic reduction capacity on hydrogen peroxide, is used as a simulated enzyme signal label, and consumes the hydrogen peroxide as an electron donor to amplify an analysis signal; by the recognition and enzyme digestion of exonuclease, the signal amplification of the mucin is further realized, so that the preparation of a photoelectrochemical sensor is completed, and the ultrasensitive and accurate detection of the mucin is realized.
Description
Technical Field
The invention relates to the technical field of tumor marker detection technology, multi-branch hybrid chain signal amplification technology and composite nano materials, in particular to a photoelectric chemical sensor for detecting mucin, which is constructed based on the catalytic performance of enzyme-like enzymes of nano materials.
Background
In recent years, the incidence of cancer has increased dramatically worldwide, which greatly threatens human health, and early detection of cancer is attracting more and more attention. Mucin is a high molecular weight and high glycosylation protein, is present in various malignant tumors, is frequently highly expressed in various cancers such as breast cancer, ovarian cancer, prostate cancer, pancreatic cancer and the like, and is a potential cancer biomarker, so that mucin has attracted great interest to scientists in the field of early diagnosis of cancers. To date, several detection methods like enzyme-linked immunosorbent assay, surface plasmon resonance, liquid chromatography-mass spectrometry and electrochemical analysis have been developed and used for the detection of mucin. However, these methods require complicated sample pretreatment and expensive instruments, and are time-consuming and labor-consuming, so that it is of great importance to develop an analytical method which is easy to operate and low in cost and can realize ultrasensitive detection of mucin.
Titanium dioxide (TiO)2) As an n-type photoelectric material, it is widely used in a Photoelectrochemical (PEC) sensor due to its non-toxicity, good catalytic activity, good biocompatibility, and unique optical and electrical properties. The key to improving the sensitivity of a PEC sensor is to improve the PEC response of the photovoltaic material. By adding zinc indium sulfide (ZnIn)2S4) With TiO2The sensitized structure formed by recombination can reduce the recombination rate of electrons and holes and effectively enhance photoelectric signals. Manganese dioxide particles (MnO) synthesized by using bovine serum albumin2BSA) has good enzyme-like catalytic activity and biocompatibility, and can regulate the electron propagation between a working electrode and an electrolyte solution by utilizing the characteristics of the BSA, thereby regulating a photoelectric signal and realizing the quantitative detection of a target.
In recent years, a signal amplification technique of a multi-branch hybrid strand has attracted much attention, and a longer DNA double helix structure with multiple branches can be obtained by a multi-branch hybrid strand reaction. MnO beneficial to loading more2/BSA, we use MnO2/BSA for H2O2The protein has good catalytic reduction capability, is marked on a multi-branch hybrid chain and is used as a novel mimic enzyme signal label, so that the PEC response of the photoelectric material can be greatly enhanced, and the sensitivity of analysis and detection is improved.
Disclosure of Invention
The invention aims to prepare a composite material with large specific surface area, good biocompatibility and photoelectric propertyOf TiO 22/ZnIn2S4Photoelectric material, which obtains a great PEC photocurrent response, a hairpin DNA chain is fixed on the surface of the photoelectric material, the hairpin DNA chain can be specifically combined with an aptamer chain (Apt) of mucin, and the aptamer chain is connected and modified with MnO2The multi-branched hybrid chains of/BSA particles are supported on the electrode surface because of MnO2Enzyme-like catalysis of/BSA, catalysis of Electron Donor H2O2Decomposing to obtain a decaying PEC signal, combining with the aptamer chain when the target is close to the electrode surface, and modifying with MnO2The multi-branch hybrid chain of the/BSA falls off, the catalytic action is weakened, the PEC current is enhanced again, the quantitative detection of the target object is realized through the change value of the PEC current, the false positive signal can be effectively avoided, and the background signal is reduced. In addition, signal amplification of a target object is realized through high selective recognition and enzyme digestion of exonuclease I (Exo I), and ultra-sensitive detection of mucin is realized.
In order to solve the technical problem, the invention is realized by the following measures:
(1) depositing a layer of gold (Au) nanoparticles on the ITO electrode: firstly, sequentially cleaning an ITO conductive electrode in acetone, ethanol and secondary water for 10 min respectively, inserting the cleaned electrode into a deposition solution containing 1-5% chloroauric acid, depositing gold nanoparticles on the surface of the ITO conductive electrode by using a potential dissolution analysis method under the condition that the deposition potential is-0.2V and the deposition time is 30-60 s, cleaning the surface of the electrode by using secondary water after deposition is finished, and naturally drying at room temperature to obtain an ITO/Au electrode;
(2) obtaining ITO/Au electrode surface growth TiO by using in-situ growth method in step (1)2And (3) particle: the ITO/Au electrode is inserted into a 25 mL beaker containing 10-15 mL of mixed solution of ammonium fluotitanate and boric acid, the concentration of the ammonium fluotitanate is 75 mM, the concentration of the boric acid is 0.2-0.4M, the reaction is carried out for 6-12 h at room temperature, after the reaction is finished, the surface of the electrode is washed by secondary water and dried for 3 h at 60 ℃, and ITO/Au/TiO is obtained2An electrode;
(3) obtaining ITO/Au/TiO in step (2) by hydrothermal method2ZnIn is grown on the surface of the electrode2S4Nano flower: mixing ITO/Au/TiO2The electrode was inserted into a 25 mL autoclave containing 10-15 mL of a mixture of zinc sulfate, indium chloride and thioacetamide to subject the mixture to ITO/Au/TiO2The electrode leans against the inner wall of the autoclave obliquely, the conductive surface faces downwards, the concentrations of zinc sulfate, indium chloride and thioacetamide are 25-100 mM and the molar ratio is 1:2:4, the mixture is heated at 90-120 ℃ for 1-3 h, the surface of the electrode is washed by secondary water after being cooled to room temperature, and the mixture is dried at 60 ℃ for 3 h to obtain ITO/Au/TiO2/ZnIn2S4An electrode;
(4) synthesis of MnO2BSA particles: weighing 250 mg of BSA, dispersing in 7 mL of secondary water, dissolving 30-50 mg of potassium permanganate in 3mL of secondary water, mixing and stirring the two solutions uniformly, transferring the two solutions into a three-neck flask, heating in a water bath to 37 ℃, keeping the temperature for 2 hours, dialyzing for 24 hours after the reaction is finished, and freeze-drying to obtain MnO2BSA particles;
(5) aptamer-ligated hybrid strand reaction: dissolving an aptamer chain Apt and a priming chain S1 in a hybridization chain reaction buffer solution (TM), wherein the obtained concentrations of Apt and S1 are both 5 mu M, dissolving a hairpin DNA molecule 1 (H1) and a hairpin DNA molecule 2 (H2) in the TM, the obtained concentrations of H1 and H2 are both 20 mu M, the TM is pH 8.0, the concentration is 20 mM, and the pH is 50 mM MgCl2Heating the chain at 95 ℃ for 10 min by using a PTC-200 thermocycler, cooling to 4 ℃ in 30S, adding a mixed solution of 100 mu L Apt and S1 into 1 mL of a mixed solution of H1 and H2 to enable the final concentrations of Apt, S1, H1 and H2 to be 0.5 mu M, 10 mu M and 10 mu M respectively, and reacting the obtained hybridization mixture at 37 ℃ for 12H to obtain an aptamer-connected multi-branch hybrid chain;
(6) synthesized and modified with MnO2Multibranched hybrid chains of/BSA particles: 500 μ L of 1 μ M MnO2The 500. mu.L of aptamer-linked multi-branched hybrid synthesized in step (5), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) were dissolved in Phosphate Buffered Saline (PBS) at pH7.4 at concentrations of 20 mM and 10 mM for EDC and NHS, respectively, and the PBS contained 55 mM phosphate, 150 mM sodium chloride and 20 mM sodium chloridePlacing the mixed solution in a dark place, shaking overnight, and centrifuging and washing with PBS (pH 7.4) to obtain modified MnO2Multibranched hybrid chains of/BSA particles;
(7) construction of the photoelectrochemical sensor: dripping 20 mu L of 0.05 mg/mL chitosan acetic acid solution into the ITO/Au/TiO solution obtained in the step (3)2/ZnIn2S4Incubating the surface of the electrode at room temperature for 4 h, wherein the concentration of the acetic acid solution is 1%, washing the surface of the electrode with PBS (pH 7.4) to remove excessive chitosan, and then washing the surface of the electrode with ITO/Au/TiO2/ZnIn2S4Inserting an electrode into a solution containing 2.5% glutaraldehyde for 2 hours, washing with PBS (pH 7.4), dripping 20 mu L of 0.5 mu M hairpin chain HP1 on the surface of the electrode, incubating at room temperature for 1 hour, washing with PBS (pH 7.4) to remove unreacted HP1, continuously dripping 20 mu L of 1% bovine serum albumin for blocking non-specific binding sites, washing with PBS (pH 7.4), dripping 20 mu L of target detection object solution with different concentrations and containing 30U ExoI on the surface of a working electrode, incubating for 1 hour under the condition of 37 ℃, and then washing the surface of the electrode with PBS (pH 7.4) to finish the construction of the photoelectrochemical immunosensor;
(8) modified ITO/Au/TiO by adopting time-current curve method2/ZnIn2S4Photoelectric chemical signal detection is carried out in a three-electrode system consisting of an electrode, an Ag/AgCl reference electrode and a platinum counter electrode under the voltage of 0.0V, and the used detection solution is 5 mL and contains 0.01M H2O2The pH of the phosphate buffer solution of 7.4, the detection of the mucin is realized by drawing a standard curve of the photoelectric signal intensity and the mucin concentration.
The invention has the beneficial effects that:
(1) the titanium dioxide/sulfur indium zinc sensitized material synthesized by the simple in-situ synthesis and hydrothermal method can effectively enhance photoelectric signals, has a large surface area, provides a good platform for fixing a large number of hairpin DNA chains, and is beneficial to amplifying detection signals and enhancing the sensitivity of analysis.
(2) By using the aptamer technology, the specific recognition of the target MUC1 can be realized; by using exonuclease, the aptamer in the aptamer/protein can be identified and enzyme-digested with high selectivity, so that the target mucin can be recycled, and the signal can be further amplified.
(3) Longer multi-branch hybrid chains obtained by using multi-branch hybrid chain reaction, wherein a plurality of branches are loaded with MnO2the/BSA particles provide a large number of active sites which can catalyze the decomposition of more electron donor H2O2Production of O2And the anode photocurrent is reduced.
(4) By utilizing a multiple signal amplification technology, an ultra-sensitive photoelectrochemical sensor for detecting the mucin is constructed, the mucin can be simply, quickly and accurately detected, and the method has great significance in early detection, metastasis and treatment of clinical cancers.
The specific implementation mode is as follows:
in order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1: application of ultrasensitive photoelectrochemical sensor in detection of mucin 1 (MUC 1)
(1) Depositing a layer of Au nanoparticles on the ITO electrode: firstly, sequentially cleaning an ITO conductive electrode in acetone, ethanol and secondary water for 10 min respectively, inserting the cleaned electrode into a deposition solution containing 1% chloroauric acid, depositing Au nanoparticles on the surface of the ITO conductive electrode by using a potential dissolution analysis method at a deposition potential of-0.2V for 60 s, cleaning the surface of the electrode by using the secondary water after the deposition is finished, and naturally drying at room temperature to obtain the ITO/Au electrode;
(2) obtaining ITO/Au electrode surface growth TiO by using in-situ growth method in step (1)2And (3) particle: the ITO/Au electrode is inserted into a 25 mL beaker containing 15 mL of mixed solution of ammonium fluotitanate and boric acid, the concentration of the ammonium fluotitanate is 75 mM, the concentration of the boric acid is 0.3M, the reaction is carried out for 9 h at room temperature, after the reaction is finished, the surface of the electrode is washed by secondary water and dried for 3 h at 60 ℃, and ITO/Au/TiO is obtained2An electrode;
(3) obtaining ITO/Au/TiO in step (2) by hydrothermal method2ZnIn is grown on the surface of the electrode2S4Nano flower: mixing ITO/Au/TiO2The electrode was inserted into a 25 mL autoclave containing 12 mL of a mixture of zinc sulfate, indium chloride and thioacetamide to subject the mixture to ITO/Au/TiO2The electrode leans against the inner wall of the autoclave obliquely, the conductive surface faces downwards, the concentrations of zinc sulfate, indium chloride and thioacetamide are respectively 25 mM, 50 mM and 100 mM, the mixture is heated at 120 ℃ for 1.5 h, the surface of the electrode is washed by secondary water after the mixture is cooled to room temperature, and the mixture is dried at 60 ℃ for 3 h to obtain ITO/Au/TiO2/ZnIn2S4An electrode;
(4) synthesis of MnO2BSA particles: weighing 250 mg of BSA, dispersing in 7 mL of secondary water, dissolving 40 mg of potassium permanganate in 3mL of secondary water, mixing and stirring the two solutions uniformly, transferring the two solutions into a three-neck flask, heating the two solutions in a water bath to 37 ℃, keeping the temperature for 2 hours, dialyzing the solution for 24 hours after the reaction is finished, and freeze-drying the solution to obtain brown MnO2BSA particles;
(5) aptamer-ligated hybrid strand reaction: dissolving an aptamer chain Apt and an initiation chain S1 in a TM, wherein the obtained Apt and S1 are both 5 mu M, dissolving H1 and H2 in the TM, the obtained H1 and H2 are both 20 mu M, heating the chain at 95 ℃ for 10 min by using a PTC-200 thermal cycler, cooling to 4 ℃ in 30S, then adding a mixed solution of 100 mu L Apt and S1 into 1 mL of a mixed solution of H1 and H2 to enable the final concentrations of Apt, S1, H1 and H2 to be 0.5 mu M, 10 mu M and 10 mu M respectively, and reacting the obtained hybridization mixture at 37 ℃ for 12H to obtain a multi-branch hybrid chain connected with the aptamer;
(6) synthesized and modified with MnO2Multibranched hybrid chains of/BSA particles: 500 μ L of 1 μ M MnO2500 μ L of the aptamer-linked multibranched hybrid synthesized in step (5), EDC and NHS were dissolved in PBS at pH7.4, wherein the concentration of EDC and NHS were 20 mM and 10 mM, respectively, and the mixture was shaken overnight in the dark and washed with PBS by centrifugation to obtain a mixture modified with MnO2Multibranched hybrid chains of/BSA particles;
(7) construction of the photoelectrochemical sensor: dripping 10 mu L of 0.05 mg/mL chitosan acetic acid solution into the ITO/Au/T obtained in the step (3)iO2/ZnIn2S4Incubating the surface of the electrode at room temperature for 4 h, wherein the concentration of the acetic acid solution is 1%, washing the surface of the electrode with PBS (pH 7.4) to remove excessive chitosan, and then adding ITO/Au/TiO2/ZnIn2S4Inserting the membrane into a solution containing 2.5% glutaraldehyde for 2 hours, washing the membrane by using PBS (pH 7.4), dripping 20 mu L of 0.5 mu M HP1 on the surface of an electrode, incubating the membrane at room temperature for 1 hour, washing the membrane by using PBS (pH 7.4) to remove unreacted HP1, continuously dripping 20 mu L of 1% bovine serum albumin for blocking non-specific binding sites, washing the membrane by using PBS (pH 7.4), dripping 20 mu L of MUC1 solution with different concentrations and containing 30U Exo I on the surface of a working electrode, incubating the membrane for 1 hour under the condition of 37 ℃ and then washing the surface of the electrode by using PBS (pH 7.4) to finish the construction of the photoelectrochemical immunosensor;
(8) modified ITO/Au/TiO by adopting time-current curve method2/ZnIn2S4Photoelectric chemical signal detection is carried out in a three-electrode system consisting of an electrode, an Ag/AgCl reference electrode and a platinum counter electrode under the voltage of 0.0V, and the used detection solution is 5 mL and contains 0.01M H2O2The detection of MUC1 was achieved by plotting the intensity of the photoelectric signal against the concentration of MUC1 in PBS (pH 7.4).
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Claims (1)
1. A preparation method of a high-sensitivity photoelectric chemical sensor for detecting mucin is characterized by comprising the following steps:
(1) depositing a layer of gold (Au) nanoparticles on an Indium Tin Oxide (ITO) conductive electrode: firstly, sequentially cleaning an ITO conductive electrode in acetone, ethanol and secondary water for 10 min respectively, inserting the cleaned electrode into a deposition solution containing 1-5% chloroauric acid, depositing gold nanoparticles on the surface of the ITO conductive electrode by using a potential dissolution analysis method under the condition that the deposition potential is-0.2V and the deposition time is 30-60 s, cleaning the surface of the electrode by using secondary water after deposition is finished, and naturally drying at room temperature to obtain an ITO/Au electrode;
(2) growing TiO on the surface of the electrode obtained in the step (1) by using an in-situ growth method2And (3) particle: the ITO/Au electrode is inserted into a 25 mL beaker containing 10-15 mL of mixed solution of ammonium fluotitanate and boric acid, the concentration of the ammonium fluotitanate is 75 mM, the concentration of the boric acid is 0.2-0.4M, the reaction is carried out for 6-12 h at room temperature, after the reaction is finished, the surface of the electrode is washed by secondary water and dried for 3 h at 60 ℃, and ITO/Au/TiO is obtained2An electrode;
(3) obtaining ITO/Au/TiO in step (2) by hydrothermal method2ZnIn is grown on the surface of the electrode2S4Nano flower: mixing ITO/Au/TiO2The electrode was inserted into a 25 mL autoclave containing 10-15 mL of a mixture of zinc sulfate, indium chloride and thioacetamide to subject the mixture to ITO/Au/TiO2The electrode leans against the inner wall of the autoclave obliquely, the conductive surface faces downwards, the concentrations of zinc sulfate, indium chloride and thioacetamide are 25-100 mM and the molar ratio is 1:2:4, the mixture is heated at 90-120 ℃ for 1-3 h, the surface of the electrode is washed by secondary water after being cooled to room temperature, and the mixture is dried at 60 ℃ for 3 h to obtain ITO/Au/TiO2/ZnIn2S4An electrode;
(4) synthesis of manganese dioxide/bovine serum Albumin (MnO)2BSA) particles: weighing 250 mg of BSA, dispersing in 7 mL of secondary water, dissolving 30-50 mg of potassium permanganate in 3mL of secondary water, mixing and stirring the two solutions uniformly, transferring the two solutions into a three-neck flask, heating in a water bath to 37 ℃, keeping the temperature for 2 hours, dialyzing for 24 hours after the reaction is finished, and freeze-drying to obtain MnO2BSA particles;
(5) aptamer-ligated hybrid strand reaction: dissolving an aptamer chain (Apt) and a priming chain (S1) in a hybridization chain reaction buffer solution (TM) to obtain both Apt and S1 concentrations of 5 mu M, dissolving a hairpin DNA molecule 1 (H1) and a hairpin DNA molecule 2 (H2) in the TM to obtain both H1 and H2 concentrations of 20 mu MTM of (2) was pH 8.0, concentration 20 mM and contained 50 mM MgCl2Heating the chain at 95 ℃ for 10 min by using a PTC-200 thermocycler, cooling to 4 ℃ in 30S, adding a mixed solution of 100 mu L Apt and S1 into 1 mL of a mixed solution of H1 and H2 to enable the final concentrations of Apt, S1, H1 and H2 to be 0.5 mu M, 10 mu M and 10 mu M respectively, and reacting the obtained hybridization mixture at 37 ℃ for 12H to obtain an aptamer-connected multi-branch hybrid chain;
(6) synthesized and modified with MnO2Multibranched hybrid chains of/BSA particles: 500 μ L of 1 μ M MnO2The BSA solution, 500. mu.L of the aptamer-linked multi-branched hybrid chain synthesized in step (5), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were dissolved in Phosphate Buffered Saline (PBS) at pH7.4, wherein the concentrations of EDC and NHS were 20 mM and 10 mM, respectively, and the PBS contained 55 mM phosphate, 150 mM sodium chloride, and 20 mM ethylenediaminetetraacetic acid, and the above-mentioned mixed solution was shaken overnight in the dark and then washed by centrifugation with PBS at pH7.4 to obtain a modified MnO2Multibranched hybrid chains of/BSA particles;
(7) construction of the photoelectrochemical sensor: dripping 20 mu L of 0.05 mg/mL chitosan acetic acid solution into the ITO/Au/TiO solution obtained in the step (3)2/ZnIn2S4Incubating the surface of the electrode at room temperature for 4 h, wherein the concentration of the acetic acid solution is 1%, washing the surface of the electrode with PBS (pH 7.4) to remove excessive chitosan, and then washing the surface of the electrode with ITO/Au/TiO2/ZnIn2S4Inserting an electrode into a solution containing 2.5% glutaraldehyde for 2 hours, washing with PBS (pH 7.4), dripping 20 mu L of 0.5 mu M hairpin chain HP1 on the surface of the electrode, incubating for 1 hour at room temperature, washing with PBS (pH 7.4) to remove unreacted HP1, continuously dripping 20 mu L of 1% bovine serum albumin for blocking non-specific binding sites, washing with PBS (pH 7.4), dripping 20 mu L of target detection substance solution with different concentrations and containing 30U of exonuclease I on the surface of a working electrode, incubating for 1 hour at 37 ℃, and then washing the surface of the electrode with PBS (pH 7.4) to finish the construction of the photoelectrochemical immunosensor;
(8) using time-current curvesThe method is to use the modified ITO/Au/TiO2/ZnIn2S4Photoelectric chemical signal detection is carried out in a three-electrode system consisting of an electrode, an Ag/AgCl reference electrode and a platinum counter electrode under the voltage of 0.0V, and the used detection solution is 5 mL and contains 0.01M H2O2The pH of the phosphate buffer solution of 7.4, the detection of the mucin is realized by drawing a standard curve of the photoelectric signal intensity and the mucin concentration.
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