CN112444546B - Electrochemical luminescence sensor for detecting oligonucleotide and preparation method thereof - Google Patents
Electrochemical luminescence sensor for detecting oligonucleotide and preparation method thereof Download PDFInfo
- Publication number
- CN112444546B CN112444546B CN201910829153.XA CN201910829153A CN112444546B CN 112444546 B CN112444546 B CN 112444546B CN 201910829153 A CN201910829153 A CN 201910829153A CN 112444546 B CN112444546 B CN 112444546B
- Authority
- CN
- China
- Prior art keywords
- ssdna
- msns
- oligonucleotide
- dna
- product dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/4163—Systems checking the operation of, or calibrating, the measuring apparatus
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Electrochemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an electrochemiluminescence sensor for detecting oligonucleotide and a preparation method thereof, relating to the technical field of DNA nanometer, the method comprises the steps of preparing mesoporous silica, loading bipyridyl ruthenium in pores of the mesoporous silica, and closing pores of the mesoporous silica by using ssDNA to obtain ssDNA-Ru-MSNs; and mixing the ssDNA-Ru-MSNs and the Product DNA, and then modifying the mixture on an electrode to obtain the oligonucleotide electrochemiluminescence sensor, wherein the ssDNA and the Product DNA are mutually complementary chains. The electrochemical luminescence sensor comprises an electrode, wherein a ssDNA-Ru-MSNs and Product DNA specific conjugate is modified on the electrode, and the ssDNA and the Product DNA are mutually complementary chains. The invention can carry out ultra-trace detection on a complex sample and is not easy to be interfered by external environment.
Description
Technical Field
The invention relates to the technical field of DNA nanometer, in particular to an electrochemiluminescence sensor for detecting oligonucleotide and a preparation method thereof.
Background
Electrochemiluminescence (ECL) is a process of triggering chemiluminescence by an electrochemical method, combines the advantages of electrochemistry and chemiluminescence, and has the characteristics of high stability, high sensitivity, controllable experimental system, wide detection range, short detection time and the like. The ECL is based on the principle that a voltage is applied to a reactant and electrochemical regeneration is performed on an electrode, the generated regenerated substance reacts with a substrate in a system to form an excited substance, energy relaxation is performed, the excited substance releases a photon to reach a ground state, and light radiation generated from the excited state to the ground state can be detected through a detector.
The ECL sensor is often combined with a biosensor to form an ECL sensor, which not only retains the advantages of good sensitivity and wide dynamic concentration response range of a Chemiluminescence (CL) sensor, but also has the advantages that the CL sensor does not have: (1) the CL reaction progress can be controlled and the selectivity can be improved by controlling the potential; (2) the generation of light near the electrodes provides improved spatial control for sensitive detection; (3) the target molecules are electrochemically modified to form a substance with CL activity and expand the analytical application thereof.
Although there are many advantages of the electrochemiluminescence sensor, the following problems still exist in detecting complex samples of biological tissues, saliva, body fluids, etc.: the electrochemiluminescence beacon substance has low light efficiency, is easily influenced by the environment (pH, temperature and the like), and is difficult to detect ultra-trace samples.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an electrochemiluminescence sensor for detecting oligonucleotide and a preparation method thereof, which can carry out ultra-trace detection on a complex sample and are not easily interfered by external environment.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:
a method of making an electrochemiluminescence sensor for detecting oligonucleotides, comprising the steps of:
s1, preparing mesoporous silica, loading bipyridine ruthenium in pores of the mesoporous silica, and sealing pores of the mesoporous silica by using ssDNA to obtain ssDNA-Ru-MSNs;
s2, carrying out isothermal amplification on the oligonucleotide to be detected to obtain Product DNA, mixing ssDNA-Ru-MSNs and the Product DNA to prepare a specific conjugate, and modifying the specific conjugate on a glassy carbon electrode to obtain the oligonucleotide electrochemiluminescence sensor, wherein the ssDNA and the Product DNA are mutually complementary chains.
Further, isothermal amplification of the oligonucleotide to be detected comprises the following steps: adding template DNA and oligonucleotide to be detected into isothermal amplification buffer solution, and then adding cutting endonuclease, polymerase and deoxyribonucleoside triphosphate for reaction to obtain Product DNA.
Further, the preparation of specific binders by mixing the ssDNA-Ru-MSNs and the Product DNA comprises the following steps: mixing 1 part of Product DNA with the concentration of 1uM-1fM and 3-8 parts of ssDNA-Ru-MSNs with the concentration of 1mg/mL according to the volume ratio, wherein the pH value of a reaction system is 6.6-8.2, the temperature is 30-37 ℃, and the reaction time is 6-24 hours.
Further, the pH value of the reaction system is 7.4, and the reaction time is 12 h.
Furthermore, the diameter of the mesoporous silica is 93-104 nm, and the diameter of the surface pores is 3.5-4 nm.
An electrochemiluminescence sensor for detecting oligonucleotides comprises electrodes, wherein specific binders of ssDNA-Ru-MSNs and Product DNA are modified on the electrodes, and the ssDNA and the Product DNA are mutually complementary chains.
Further, the electrode is a glassy carbon electrode.
Further, the electrochemical luminescence sensor also comprises detection liquid, and the detection liquid comprises TPA with the concentration of 0.01mol/L and PBS with the concentration of 0.1mol/L, pH of 7.4.
A method for detecting an oligonucleotide by an electrochemiluminescence sensor, comprising the steps of: the electrodes were inserted into the test solution for ECL measurements.
Further, the measurement voltage of the ECL measurement is 0V-1.3V.
Compared with the prior art, the invention has the advantages that:
(1) in the electrochemical luminescence sensor for detecting oligonucleotide, the mesoporous silica has pores on the surface, so that the mesoporous silica can be conveniently loadedThe signal molecule Ru seals the orifice through ssDNA, so that Ru escape is avoided, and meanwhile, the mesoporous silica has good biocompatibility and stability, and can be stably and uniformly dispersed in a detection system when in use. The Product DNA is prepared by isothermal amplification, an oligonucleotide signal to be detected can be amplified, the Product DNA and ssDNA react to form a double-chain structure and are separated from the surface of mesoporous silica, signal molecules Ru are released to generate an ECL signal, and the detection range of the electrochemical luminescence sensor is 1.0 multiplied by 10-14mol/L～1.0×10-8mol/L, detection limit of 1.885X 10- 15And the kit is mol/L (S/N is 3: 1), can perform ultra-trace detection on a complex sample, and is not easily interfered by an external environment.
Drawings
FIG. 1 is a schematic diagram of the DNA amplification technique used for rabies virus oligonucleotide detection in the example of the present invention;
FIG. 2 is a diagram showing the characterization of DNA-Ru-MSNs in the example of the present invention;
FIG. 3 is a diagram showing the pore size characterization of DNA-Ru-MSNs in the example of the present invention;
FIG. 4 is a diagram of the UV-VIS spectrophotometer and Fourier infrared spectrometer of DNA-Ru-MSNs analyzing materials in the embodiment of the invention;
FIG. 5 is an electrophoretic image of 12% polyacrylamide gel analysis of the product in the example of the present invention;
FIG. 6 is a graphical illustration of a feasibility analysis of an ECL sensor in an embodiment of the invention;
FIG. 7 shows the concentration of Target DNA at 1.0X 10 in the examples of the present invention-10An electrochemical cyclic voltammogram of Ru-MSNs/GCE at mol/L;
FIG. 8 is a graph of the effect of pH, temperature on ECL measurements in an example of the present invention;
FIG. 9 is a graph showing the specificity, reproducibility and stability analysis of ECL detection in the examples of the present invention.
Detailed Description
Embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.
Referring to fig. 1, an embodiment of the present invention provides a method for preparing an electrochemiluminescence sensor for detecting oligonucleotides, including:
s1, preparing mesoporous silica, loading bipyridyl ruthenium in pores of the mesoporous silica, and sealing pores of the mesoporous silica by using ssDNA to obtain ssDNA-Ru-MSNs, wherein the diameter of the mesoporous silica is 93-104 nm, and the diameter of the surface pores is 3.5-4 nm.
S2, preparing Product DNA, adding the template DNA and the oligonucleotide to be detected into isothermal amplification buffer solution, and then adding cutting endonuclease, polymerase and deoxyribonucleoside triphosphate for reaction to obtain the Product DNA.
S3, mixing ssDNA-Ru-MSNs and Product DNA to prepare a specific conjugate, and modifying the specific conjugate on a glassy carbon electrode to obtain the oligonucleotide electrochemiluminescence sensor, wherein the ssDNA and the Product DNA are mutually complementary chains.
Wherein, the specific conjugate of the ssDNA-Ru-MSNs and the Product DNA is prepared by the following method: mixing 1 part of Product DNA with the concentration of 1uM-1fM and 3-8 parts of ssDNA-Ru-MSNs with the concentration of 1mg/mL according to the volume ratio, wherein the pH value of a reaction system is 6.6-8.2, the temperature is 30-37 ℃, and the reaction time is 6-24 h.
The invention also provides an electrochemiluminescence sensor for detecting the oligonucleotide, which comprises an electrode and detection liquid, wherein the electrode in the embodiment adopts a glassy carbon electrode, the electrode is modified with a ssDNA-Ru-MSNs and Product DNA specific conjugate, and the ssDNA and the Product DNA are mutually complementary chains; the detection solution comprises TPA with the concentration of 0.01mol/L and PBS with the concentration of 0.1mol/L, pH of 7.4.
In actual use, the detection solution is prepared according to actual needs, and the concentration and the pH value of the detection solution can be adjusted according to needs.
The invention also provides a method for detecting the oligonucleotide by the electrochemical luminescence sensor, which comprises the following steps: adding the oligonucleotide to be detected into detection solution with an electrode to carry out ECL measurement, wherein the measurement voltage of the ECL measurement is 0V-1.3V.
The specific method according to the present invention will be described in detail below.
S1 synthetic mesoporous silica
0.2g of cetyltrimethyl bromide powder (CTAB) was dissolved in 96mL of ultrapure water, followed by addition of 2.5mL of an aqueous solution of sodium hydroxide having a concentration of 1.0mol/L to adjust the pH. Heating to 90 ℃, dropwise adding 1mL of TEOS (tetraethyl orthosilicate) into the solution at a constant speed under continuous stirring, vigorously stirring for reaction for 3h, centrifugally collecting the generated white precipitate, respectively washing with ultrapure water and ethanol for three times, drying for 12h under a vacuum condition at the temperature of 80 ℃, calcining for 5h under the temperature of 550 ℃ to remove a CTAB template, and obtaining white mesoporous silica (hereinafter referred to as mesoporous silica spheres or MSNs) powder.
Referring to FIG. 2A, Transmission Electron Microscopy (TEM) showed that the average diameter of the calcined MSNs was about 93nm, with a highly ordered lattice array and a uniform, well-defined mesostructure, in the form of a Ru (bpy) charge3 2+Providing a good space.
S2, loading bipyridyl ruthenium on mesoporous silicon spheres (the bipyridyl ruthenium can be replaced by a derivative of the bipyridyl ruthenium)
50mgMSNs were dispersed in 50mL of absolute ethanol and stirred until completely dissolved, and an excess of Ru (bpy) was added3 2+After stirring for 12 hours, 200. mu.L of APTES (3-aminopropyl) triethoxyslane, 3-aminopropyltriethoxysilane was added to the mixed solution, and stirring was continued for 6 hours to complete the amination on the surface of the silicon spheres.
Centrifugally collecting orange aminated mesoporous silicon spheres, washing with ethanol for three times, and vacuum drying at 60 ℃ to obtain yellow aminated mesoporous silicon spheres (NH)2-Ru-MSNs) powder.
Referring to FIG. 2B, it can be seen that MSNs are loaded with Ru (bpy)3 2+Then, the color changed from white to yellow, and the same MSNs loaded with Ru (bpy)3 2+Post-dispersion in ethanol fluoresced red, indicating Ru (bpy)3 2+Successful loading into MSNs. It can also be seen in the fluorescence spectrum that Ru (bpy)3 2+The fluorescence of (B) was identical to that of the Ru-MSNs, indicating that Ru (bpy)3 2+Good optical properties are maintained after entering MSNs.
Referring to FIG. 3A, the result is shown by N2The adsorption-desorption isotherms of (A) can indicate that the MSN is porous with a mean pore size of 3.9nm, which enables Ru (bpy)3 2+Sealing in the MSN pore channel; Brunauer-Emmett-Teller (BET) analysis showed that the specific surface area of the MSN was 456cm2In the case of Ru (bpy) loading3 2+After that, its specific surface area became 223cm2Therefore, it can be seen that the pores of the MSN are filled with a large amount of Ru (bpy)3 2+。
S3 mesoporous silicon spheres with DNA blocked amination
Weighing 1mg of NH2The Ru-MSNs were dispersed in 500. mu.L of hybridization buffer (0.01mol/L Tris-HCl, 0.05mol/L NaCl, and 0.01mol/L MgCl2pH 7.4), 25. mu.L of 10 was added-5The ssDNA is reacted for 1h at 37 ℃ in mol/L with shaking, so that the ssDNA is electrostatically adsorbed on NH2The surface of the-Ru-MSNs is washed by centrifugation, the non-adsorbed ssDNA is removed, and the obtained closed silicon spheres are stored at 4 ℃ for the next step.
Referring to FIG. 3B, MSNs are initially at-32.1 mV, mainly due to the fact that the surface of MSNs carry a large number of-OH groups, and after APTES amination modification, the-OH groups on the surface of MSNs are changed into-NH groups2The potential thereof also became +30.6 mV; after modification of DNA on MSNs, negatively charged ssDNA and positively charged NH2The surface charge of the MSNs is reduced sharply (-24.7mV) by electrostatic adsorption combination of the MSNs, and the successful synthesis of the DNA-Ru-MSNs composite material can be seen through the phenomenon of potential change.
Referring to FIG. 4A, Ru (bpy)3 2+As can be seen from the UV spectra of Ru-MSNs, the UV spectra before and after the reaction are roughly consistent, and are both Ru (bpy)3 2+Characteristic absorption wavelength of (B), Ru (bpy)3 2+Was successfully encapsulated in silicon dioxide, and Ru (bpy) remained3 2+Original optical properties.
Referring to FIG. 4B, the IR spectra of MSNs and Ru-MSNs are shown at 1100cm-1The strong peak at (A) is due to stretching vibration of Si-O-Si (Tai et al 2007), which exists before and after the reaction, and appears 560cm after the MSN amination-1、1520cm-1、1559cm-1(N-H), etc., indicating successful amination of MSNs.
S4 DNA amplification
To a concentration of 10 in 1. mu.L-9mu.L of 3. mu.L of EB buffer solution (isothermal amplification buffer solution) was added to mol/L of template DNA to obtain amplification solutions, and multiple aliquots were added at a volume of 1. mu.L and a concentration of 10. mu.L-14mol/L～10-9mol/L) different rabies virus oligonucleotides (in practice, the rabies virus oligonucleotides can be replaced by other oligonucleotides to be detected) are added into different amplification solutions, 2 muL of Nt.BbvCI (cleavage endonuclease) with the concentration of 20U, 2 muL of polymerase Klenow fragment exo- (the proteolytic Product of E.coli DNA polymerase I with the concentration of 10U, 5'→ 3' of DNA polymerase activity but 5'→ 3' of exonuclease activity is lost) and 1 muL of dNTP (deoxy-riboside triphosphate) with the concentration of 0.025mol/L are added into each solution and mixed, the mixture is placed at the temperature of 37 ℃ for reaction for 1h and then stored at the temperature of 80 ℃ for 20min for termination reaction, and DNA amplification Product DNA is obtained.
To verify the mechanism of the DNA amplification experiment, 12% polyacrylamide gel electrophoresis was used to characterize the entire course of the reaction.
Referring to FIG. 5, when Product DNA (second lane) and ssDNA (third lane) were mixed, a distinct hybridization band (fourth lane) was observed, demonstrating that Product DNA and ssDNA can be recognized and hybridized in complementary pairs. Hybridization complementation between Tempalte DNA (fifth lane) and Tagert DNA (sixth lane) was also demonstrated when they were mixed (seventh lane). In lane 8, a band corresponding to the 7 th lane is shown, and after ssDNA is added (ninth lane), a band corresponding to the Product DNA in the fourth lane hybridized with ssDNA is shown, and these results confirm that isothermal amplification of DNA produces Product DNA, and the feasibility of the amplification method is verified.
As shown in Table 1, the nucleotide sequence of the DNA used in this step is shown.
Base sequences used in Table 1
S5 preparation of electrode
Polishing a glassy carbon electrode to be used on a polishing plate by using alumina powder of 1.0 μm, 0.3 μm and 0.05 μm respectively until the electrode surface is smooth, then cleaning the electrode surface by using ultrapure water, then performing ultrasonic cleaning in nitric acid solution (nitric acid: water: 1), ethanol and pure water in sequence, and finally performing blow-drying by using nitrogen to obtain the Glassy Carbon Electrode (GCE).
10 μ L of ssDNA-NH at a concentration of 0.1mg/mL2And mixing and incubating the MSNs and 10 mu L of Product DNA, and taking out 5 mu L of Product DNA to be fixed on GCE to obtain the electrochemiluminescence biosensor.
ECL measurements were performed in PBS containing TPA at a concentration of 0.01mol/L and a concentration of 0.1mol/L, pH of 7.4 using a conventional three-electrode system to characterize the modified GCE electrodes.
The measurement voltage of the ECL is 0V-1.3V, and the scanning speed is 0.3V/s; control experiments were performed under the same conditions for Random sequences (Random DNA), base Mismatch sequences (Mismatch DNA), base deletion sequences (protected DNA), and a mixture of the three with the target sequence. All experiments were performed at room temperature and measured three times.
The feasibility of the ECL sensor was analyzed in this example, and the strongest signal is the curve corresponding to the unclosed Ru-MSNs, released Ru (bpy)3 2+A strong ECL signal is generated.
The weakest signal is the corresponding curve after addition of ssDNA, since the probe effectively encapsulates Ru (bpy) in the pores of MSNs as a blocked ssDNA probe3 2+They are prevented from reacting with TPA in the test solution, so no ECL signal is generated.
The signal is atBetween the two is the curve corresponding to the product DNA binding to ssDNA after mixing the products of the DNA amplification reaction with ssDNA-Ru-MSNs, due to the weak adhesion of the DNA duplex rigid structure to MSNs. Thus, an ECL response can be observed, indicating a portion of Ru (bpy)3 2+The molecules are released from the MSNs and react with TPA.
As shown in FIG. 7, the concentration of the DNA at Target is 1.0X 10-10And at mol/L, the electrochemical cyclic voltammetry curve of Ru-MSNs/GCE.
TPA begins to undergo oxidation at a peak potential of 0.7V. With increasing voltage, Ru (bpy)3 2+A redox reaction occurred and a distinct ECL peak was produced at 1.15V.
The reaction mechanism is as follows:
Ru(bpy)3 2+-e→Ru(bpy)3 3+…………………(1)
TPA–e→TPA+.→TPA.+H+………………(2)
Ru(bpy)3 3++TPA.→Ru(bpy)3 2+*+product……(3)
Ru(bpy)3 2+*→Ru(bpy)3 2++hγ…………………(4)
furthermore, in order to obtain the optimal performance conditions of the ECL sensor, the incubation time of the Product DNA and the ssDNA-Ru-MSNs, the pH value of the test solution and other conditions are optimized respectively. As shown in fig. 8, with the increase of the incubation time, the ECL signal of the biosensor tends to be stable after 12h, indicating that the Product DNA and the ssDNA on the surface of the silica sphere are complementarily saturated at this time; furthermore, the ECL signal of the biosensor is strongest at pH close to 7.4. Therefore, an incubation time of 12h and a pH of 7.4 are optimal conditions.
S6 rabies virus detecting oligonucleotide
The ECL signal of the biosensor was detected in 0.1mol/LPBS solution containing 0.01mol/L TPA using rabies virus oligonucleotide as a target.
Fig. 9A shows ECL signals detected by the biosensor for the target at different concentrations, and it can be seen that the ECL signal intensity gradually increases with the increase of the target concentration, indicating that the biosensor responds well to the change of the target concentration.
FIG. 9B is a standard curve of the target DNA detected by the electrochemical luminescence sensor, at 1.0X 10-14mol/L～1.0×10-8In the mol/L range, the ECL intensity is linearly related to the logarithm of the target concentration. The linear regression equation is Y-749.4116X +4316.1450 with the correlation coefficient R20.9774, wherein Y is the ECL peak intensity and X is the logarithm of the nucleotide concentration. The detection limit of the sensor is 1.885 multiplied by 10 according to calculation-15mol/L(S/N＝3：1)
S7, specificity of ECL sensors for rabies virus oligonucleotides was studied with equal amounts of Random sequence (Random DNA), deleted sequence (Detected DNA), mismatched sequence (Mismatch DNA), and mixture as interfering agents.
Referring to fig. 9C, it can be seen that: the ECL strength of the ECL sensor remained stable (3.8% change) over the 200s test, indicating that the ECL sensor had better stability.
As can be seen from FIG. 9D, only in the presence of the target DNA, the ECL value was large, and the influence of the interfering agent on the ECL intensity ratio was insignificant, indicating that the ECL sensor has good specificity.
Detection Using actual samples
In order to evaluate the value of the biosensor of the present invention in practical applications, the present example uses real saliva samples as a complex matrix for detection.
Saliva samples were analyzed by standard addition methods and the accuracy and precision of the results were assessed by Relative Standard Deviation (RSD).
The results are shown in Table 2, and the samples were spiked with 1.0X 10-12mol/L、5.0×10-12mol/L、1.0×10-11The standard target DNA in mol/L was used for recovery testing and saliva samples were evaluated and no target DNA was found in the clean samples.
After the standard target DNA is added, the recovery rate and the RSD are respectively in the ranges of 94.8% -102.3% and 1.78% -7.85%, and the ECL detection method is proved to have good accuracy and can be used for saliva sample analysis.
Table 2 measurement and recovery test results of TargetDNA in saliva.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone with the teaching of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as the present invention, are within the protection scope.
Claims (8)
1. A method for preparing an electrochemiluminescence sensor for detecting oligonucleotides, comprising: the method comprises the following steps:
s1, preparing mesoporous silica, wherein the diameter of the surface pore diameter of the mesoporous silica is 3.5-4 nm, bipyridine ruthenium is loaded in the pores of the mesoporous silica, ssDNA is used for sealing the pores of the mesoporous silica, and the ssDNA is electrostatically adsorbed on aminated mesoporous silicon spheres NH2Obtaining ssDNA-Ru-MSNs on the surface of the-Ru-MSNs;
s2, carrying out isothermal amplification on the oligonucleotide to be detected to obtain Product DNA, mixing ssDNA-Ru-MSNs and the Product DNA to prepare a specific conjugate, and modifying the specific conjugate on a glassy carbon electrode to obtain the oligonucleotide electrochemiluminescence sensor, wherein the ssDNA and the Product DNA are mutually complementary chains;
isothermal amplification of the oligonucleotide to be detected in step S2 comprises the steps of: adding template DNA and oligonucleotide to be detected into isothermal amplification buffer solution, and then adding cutting endonuclease, polymerase and deoxyribonucleoside triphosphate for reaction to obtain Product DNA;
the preparation of the specific binding substance by mixing the ssDNA-Ru-MSNs and the Product DNA in the step S2 comprises the following steps:
mixing 1 part of Product DNA with the concentration of 1uM-1fM and 3-8 parts of ssDNA-Ru-MSNs with the concentration of 1mg/mL according to the volume ratio, wherein the pH value of a reaction system is 6.6-8.2, the temperature is 30-37 ℃, and the reaction time is 6-24 hours.
2. The method of claim 1 for preparing an electrochemiluminescence sensor for detecting oligonucleotides, wherein: the pH value of the reaction system is 7.4, and the reaction time is 12 h.
3. The method of claim 1, wherein the electrochemical luminescence sensor for detecting oligonucleotide is prepared by: the diameter of the mesoporous silica is 93-104 nm.
4. An electrochemiluminescence sensor for detecting an oligonucleotide, which is manufactured by the manufacturing method of any one of claims 1 to 3, wherein: the electrode is modified with a ssDNA-Ru-MSNs and Product DNA specific conjugate, and the ssDNA and the Product DNA are mutually complementary chains.
5. The electrochemiluminescence sensor of claim 4, wherein: the electrode is a glassy carbon electrode.
6. The electrochemiluminescence sensor of claim 4, wherein: the electrochemiluminescence sensor also comprises detection liquid, wherein the detection liquid comprises TPA with the concentration of 0.01mol/L and PBS with the concentration of 0.1mol/L, pH of 7.4.
7. A method for detecting an oligonucleotide using the electrochemiluminescence sensor of any of claims 4 to 6, comprising the steps of: the electrodes were inserted into the test solution for ECL measurements.
8. The method for detecting an oligonucleotide according to claim 7, wherein: the measurement voltage of ECL measurement is 0V-1.3V.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910829153.XA CN112444546B (en) | 2019-09-03 | 2019-09-03 | Electrochemical luminescence sensor for detecting oligonucleotide and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910829153.XA CN112444546B (en) | 2019-09-03 | 2019-09-03 | Electrochemical luminescence sensor for detecting oligonucleotide and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112444546A CN112444546A (en) | 2021-03-05 |
| CN112444546B true CN112444546B (en) | 2022-07-01 |
Family
ID=74734527
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910829153.XA Active CN112444546B (en) | 2019-09-03 | 2019-09-03 | Electrochemical luminescence sensor for detecting oligonucleotide and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112444546B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108418A (en) * | 2009-12-25 | 2011-06-29 | 中国科学院广州生物医药与健康研究院 | Loop-mediated isothermal amplification detection method and kit for H1N1 influenza A viruses |
| CN102854183A (en) * | 2012-09-04 | 2013-01-02 | 上海师范大学 | Method for detecting melamine through solid-state electrochemiluminescence |
| CN110146566A (en) * | 2019-06-14 | 2019-08-20 | 西南大学 | Modified electrode, combination product and its electroluminescent chemiluminescence biosensor and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE602006012455D1 (en) * | 2005-03-03 | 2010-04-08 | Ca Nat Research Council | METHOD AND COMPOSITIONS FOR DETECTING AND ANALYZING NUCLEIC ACIDS BY MEANS OF SIGNAL GAINING |
-
2019
- 2019-09-03 CN CN201910829153.XA patent/CN112444546B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108418A (en) * | 2009-12-25 | 2011-06-29 | 中国科学院广州生物医药与健康研究院 | Loop-mediated isothermal amplification detection method and kit for H1N1 influenza A viruses |
| CN102854183A (en) * | 2012-09-04 | 2013-01-02 | 上海师范大学 | Method for detecting melamine through solid-state electrochemiluminescence |
| CN110146566A (en) * | 2019-06-14 | 2019-08-20 | 西南大学 | Modified electrode, combination product and its electroluminescent chemiluminescence biosensor and application |
Non-Patent Citations (5)
| Title |
|---|
| A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes;Roy Sharmili等;《BIOSENSORS & BIOELECTRONICS》;20160622;第86卷;摘要，材料和方法部分，图1，Supplementary information的ST 1 * |
| A triple signal amplification method for chemiluminescent detection of the cancer marker microRNA-21;Chen Donghua等;《MICROCHIMICA ACTA》;20190610;第186卷(第7期);第410-8页 * |
| Stimulus-response mesoporous silica nanoparticle-based chemiluminescence biosensor for cocaine determination;Chen Zhonghui等;《BIOSENSORS & BIOELECTRONICS》;20150806;第75卷;第8-14页 * |
| 介孔二氧化硅介导的功能核酸检测技术研究进展;李舒婷等;《生物技术通报》;20180306(第09期);第139-148页 * |
| 基于纳米介孔二氧化硅可控制系统电致化发光法检测生物分子;王爱国;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》;20120715(第07期);引言，实验部分，结果与讨论部分，示意图2-1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112444546A (en) | 2021-03-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Indium tin oxide-coated glass modified with reduced graphene oxide sheets and gold nanoparticles as disposable working electrodes for dopamine sensing in meat samples | |
| Tian et al. | A core–shell-structured molecularly imprinted polymer on upconverting nanoparticles for selective and sensitive fluorescence sensing of sulfamethazine | |
| CN108801990B (en) | CsPbBr-based3Omethoate detection method of perovskite quantum dot-molecular imprinting fluorescence sensor | |
| Zhang et al. | Sensitive electrochemical determination of rhodamine B based on a silica-pillared zirconium phosphate/nafion composite modified glassy carbon electrode | |
| CN113075269A (en) | Electrochemical luminescence aptamer sensor for specifically detecting chloramphenicol and preparation method and application thereof | |
| CN109307699A (en) | The preparation and application of electrochemical sensor based on halloysite nanotubes and graphene | |
| CN109187470B (en) | Aptamer-mediated silver-doped carbon dot catalysis H2O2Method for measuring lead by reaction with TMB (tetramethylbenzidine) through fluorescence spectrum | |
| Wang et al. | A new amperometric hydrazine sensor based on Prussian blue/single‐walled carbon nanotube nanocomposites | |
| CN109490385A (en) | Biosensor and preparation method thereof based on Au-ZIF-8/OMC mesoporous carbon | |
| CN108239286A (en) | Silanization carbon quantum dot surface caffeic acid molecularly imprinted polymer, preparation method and its application | |
| CN111175358B (en) | Preparation method of cyfluthrin molecular imprinting electrochemiluminescence sensor and method for detecting cyfluthrin | |
| Puttasakul et al. | Hydrogel based-electrochemical gas sensor for explosive material detection | |
| CN110487866A (en) | A kind of application of Porous hollow Nano carbon balls material prepared and its detect nitrite | |
| CN113340860A (en) | Manganese-doped carbon dot and Mn-CDs solution for detecting Fe3+, test paper, preparation method of test paper and detection method of test paper | |
| CN112444546B (en) | Electrochemical luminescence sensor for detecting oligonucleotide and preparation method thereof | |
| CN109187687B (en) | Preparation of conjugated organic microporous material modified electrode and application of modified electrode as peroxynitroso anion electrochemical sensor | |
| CN110596082A (en) | Probe for detecting trace uranyl ions and portable ECL detector based on probe | |
| CN109813787A (en) | A kind of MnO2/Fe2O3@amorphous carbon composite material, aptamer sensor and its preparation method and application | |
| Hosseini et al. | A novel Lu3+ fluorescent nano-chemosensor using new functionalized mesoporous structures | |
| CN105954248B (en) | Arsenite detection method based on DNA biology quantum dot enhancement effect of fluorescences | |
| CN110702760B (en) | Nano-gold-DNA (deoxyribonucleic acid) net structure electrochemical biosensor for detecting uranyl ions as well as preparation method and application of nano-gold-DNA net structure electrochemical biosensor | |
| CN110511745B (en) | Preparation method of water-soluble fluorescent silicon quantum dots and application of water-soluble fluorescent silicon quantum dots in selective detection of p-nitrophenol | |
| CN112322280A (en) | Preparation method of piperazine functionalized carbon quantum dots and application of piperazine functionalized carbon quantum dots in oxytetracycline detection | |
| Tang et al. | Polyvinylpyrrolidone as a new fluorescent sensor for nitrate ion | |
| CN111896596A (en) | Electrochemical luminescence method using covalent organic framework as co-reactant and arsenite detection application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |