CN111272742A - Electrochemiluminescence sensor based on metal organic gel composite material and metal organic framework and preparation and detection methods thereof - Google Patents

Electrochemiluminescence sensor based on metal organic gel composite material and metal organic framework and preparation and detection methods thereof Download PDF

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CN111272742A
CN111272742A CN202010153243.4A CN202010153243A CN111272742A CN 111272742 A CN111272742 A CN 111272742A CN 202010153243 A CN202010153243 A CN 202010153243A CN 111272742 A CN111272742 A CN 111272742A
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electrochemiluminescence
metal organic
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mog
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CN111272742B (en
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石康中
毛昌杰
张怡文
柳星培
陈京帅
牛和林
金葆康
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Anhui University
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Anhui University
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    • 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

Abstract

The invention discloses an electrochemiluminescence sensor based on a metal organic gel composite material and a metal organic framework and a preparation method and a detection method thereof&g‑C3N4The method comprises the following steps of @ Zr-MOG composite nano material, fixing capture DNA modified by depurination/pyrimidine sites on the surface of the composite nano material through gold-sulfur bonds; the other end of the capture DNA is connected with a quencher Fe-MIL-88 metal organic framework through an amide bond. The invention realizes the detection of the target RNA sequence through the electrochemiluminescence sensor, and has the advantages of simple method, wide detection range, high sensitivity and easy operation.

Description

Electrochemiluminescence sensor based on metal organic gel composite material and metal organic framework and preparation and detection methods thereof
Technical Field
The invention belongs to the technical field of novel functional materials and biosensing, and particularly relates to an electrochemiluminescence sensor based on a metal organic gel composite material and a metal organic framework, and a preparation method and a detection method thereof.
Background
In recent years, Metal Organogels (MOGs) have attracted increasing attention from researchers as an important branch of supramolecular chemistry. MOG is a porous, soft material formed by introducing metal ions into supramolecular organogels, which combines the advantages of supramolecular gels and metal-organic frameworks. The introduction of different metal ions and ligands brings about a number of unique properties to the gel system, such as electrical conductivity, magnetism, fluorescence, etc. (Santos-Lorenzo, J.san Jos re-Velado, RubenAllo, Jonathan Beobide, Garikoitz
Figure BDA0002403166320000011
Pedro Castillo,Oscar Luque,Antonio
Figure BDA0002403166320000012
Sonia, et al 2019 Microporous Mesoporous Mat.,284: 128-132.). These properties fulfill specific functions that make them potentially useful in the fields of catalysis, adsorption, and detection. MOG is a good medium, its exposed groups and porous structure enable easy combination of chemiluminescent and modifying materials, and its good biocompatibility makes it a unique advantage in biological monitoring. However, there are few reports of using MOG in ECL systems.
Metal Organic Frameworks (MOFs) are a new class of porous organic-inorganic hybrid materials. In addition to the advantages of having the same exposed groups and easily modified organic ligands and variable metal nodes available for functionalization, their rigid backbone, clear geometry and single particle properties make them uniquely advantageous in electrochemical sensors compared to MOG. The MIL series is one of the important branches of MOFs. The exposed amino and carboxyl of Fe-MIL-88 are easy to combine with the modified DNA, and a wide ultraviolet absorption peak creates good conditions for receiving energy. These desirable properties indicate that metal organic framework materials are expected to be extended to many other optical and electrical related fields.
In recent years, scientists have performed laborious work on early detection of diseases, such as reverse transcription polymerase chain reaction (RT-PCR) and antibody-based detection, such as enzyme-linked immunosorbent assay (ELISA), but have drawbacks of long time consumption, difficult operation, and the like. Therefore, the development of a simple, accurate, highly sensitive and reliable biomedical analysis and diagnosis method is of great importance for the detection of diseases. Electrochemiluminescence (ECL) is an optical and electrochemical combined detection method, and can significantly improve the signal-to-noise ratio and sensitivity because an external light source is not required to be added, allow the control of luminescence in time, space and electrode potential, and avoid electrochemical interference. Therefore, the application of electrochemiluminescence technology to detection has its unique advantages.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art, and provides an electrochemiluminescence sensor based on a metal organic gel composite material and a metal organic framework, and a preparation method and a detection method thereof, so that the accurate detection of a target RNA sequence can be realized.
In order to realize the purpose of the invention, the following technical scheme is adopted:
the invention firstly discloses an electrochemiluminescence sensor based on a metal organic gel composite material and a metal organic framework, wherein AuNPs are fixed on the surface of a Glassy Carbon Electrode (GCE)&g-C3N4@ Zr-MOG composite nanomaterial, in which AuNPs&g-C3N4Fixing capture DNA modified by depurination/pyrimidine (AP) sites on the surface of the @ Zr-MOG composite nano material through a gold-sulfur bond; the other end of the capture DNA modified by the depurination/pyrimidine site is connected with a quencher Fe-MIL-88 metal organic framework through an amido bond; the sequence of the capture DNA is complementary with the base of the sequence of the RNA to be detected.
The principle of detecting the target RNA sequence by the electrochemiluminescence sensor provided by the invention is as follows: the AuNPs&g-C3N4The @ Zr-MOG composite nano material and the quencher Fe-MIL-88 metal organic framework are linked through capture DNA modified by depurination/pyrimidine sites, so that the electrochemiluminescence signal is quenched;when an RNA sequence to be detected is dripped on the electrochemiluminescence sensor, capturing DNA and the base of the RNA sequence to be detected are complementarily paired, and then a specific cutting site of endonuclease IV is added to remove the cut capturing DNA segment and a quencher Fe-MIL-88 metal organic framework connected with the cut capturing DNA segment, so that the electrochemiluminescence signal is gradually recovered, and the electrochemiluminescence sensor to be detected is obtained; and testing the electrochemiluminescence signal of the electrochemiluminescence sensor to be tested to obtain the electrochemiluminescence signal intensity of the RNA sequence to be tested, and judging the concentration of the RNA sequence sample to be tested by utilizing a standard relation curve of the electrochemiluminescence signal intensity and the concentration of the RNA sequence to be tested.
The endonuclease IV is an apurinic/pyrimidine (AP) endonuclease, which hydrolyzes the complete AP site on double-stranded DNA and cleaves the first phosphodiester bond at the 5' end, which is connected to the AP site.
Further, the AuNPs&g-C3N4The @ Zr-MOG composite nano material is doped with g-C3N4Au nano-particles are uniformly loaded on the Zr metal organic gel through electrostatic adsorption.
The preparation method of the electrochemiluminescence sensor comprises the following steps:
step 1, sequentially putting glassy carbon electrodes on chamois and using Al with the diameter of 0.3 mu m2O3Powder, Al 0.05 μm in diameter on fleece2O3Polishing the powder until the surface is mirror-like, then washing with deionized water, and then ultrasonically cleaning with deionized water, ethanol and deionized water in sequence to remove Al on the surface2O3And finally with N2Drying;
10 μ L of AuNPs&g-C3N4The @ Zr-MOG dispersed liquid is dripped on the surface of the glassy carbon electrode after treatment and is dried at room temperature;
step 2, connecting the capture DNA modified by the depurination/pyrimidine site with a quencher Fe-MIL-88 metal organic framework through an amido bond to obtain a dispersion liquid of the Fe-MIL-88 capture DNA;
to 15. mu.L of the dispersion of Fe-MIL-88-capture DNA was added 0.6. mu.L of 10mmol/LTris (2-carboxyethyl) phosphine aqueous solution, treated at room temperature for 1 hour to cleave S-S bond, then dropped on the surface of glassy carbon electrode, incubated overnight at 4 ℃, washed with 10mmol/L, pH ═ 8.0 TE buffer solution, and finally washed with N2Drying;
and 3, dropwise adding 15 mu L of phosphate buffer solution containing 1mmol/L of 6-mercaptohexanol for blocking unreacted active sites onto the surface of the glassy carbon electrode, treating at normal temperature for 1 hour, and then washing with 10mmol/L, pH-8.0 TE buffer solution to remove unreacted 6-mercaptohexanol, so as to obtain the electrochemiluminescence sensor based on the metal organic gel composite material and the metal organic framework, wherein the electrochemiluminescence sensor is used for detecting target RNA.
Further, the AuNPs&g-C3N4The @ Zr-MOG dispersion was prepared as follows:
step 1, g-C3N4Synthesis of @ Zr-MOG Dispersion
Firstly, 3.0mL of g-C with the concentration of 1mg/mL3N4The solution was mixed with 7.0mL of N, N-dimethylformamide and transferred to a reagent bottle; then adding 0.105g of trimesic acid, carrying out ultrasonic treatment for 20 minutes, then adding 0.1165g of anhydrous zirconium chloride, and continuing ultrasonic treatment for 30 minutes to form a uniform solution; heating the obtained homogeneous solution at 120 deg.C for 9 hr to obtain g-C in a reagent bottle3N4@ Zr-MOG cylindrical gel; the obtained g-C3N4Freeze-drying the @ Zr-MOG cylindrical gel at-114 deg.C for 2 days to obtain gel powder;
grinding 20mg of gel powder, dispersing into 20mL of deionized water, and carrying out ultrasonic treatment for 4 hours to obtain g-C3N4@ Zr-MOG dispersion;
step 2, synthesis of AuNPs dispersion liquid
In ice bath, adding 0.6mL of 0.1mol/L sodium borohydride solution into 20mL of 250mmol/L chloroauric acid solution, stirring for 10 minutes, reacting the obtained mixture at room temperature for 3 hours to obtain gold nanoparticle AuNPs dispersion, and storing at 4 ℃ for later use;
step 3, AuNPs&g-C3N4Preparation of a @ Zr-MOG Dispersion
To 200. mu.L of deionized water were added 100. mu.L of the AuNPs dispersion obtained in step 2 and 300. mu.L of g-C obtained in step 13N4The @ Zr-MOG dispersion is treated by ultrasonic for 2 hours to obtain AuNPs&g-C3N4@ Zr-MOG dispersion.
Further, the method for connecting the capture DNA modified by the depurination/pyrimidine site with the quencher Fe-MIL-88 metal organic framework through an amide bond is as follows:
step 1, dissolving 0.126g of 2-aminoterephthalic acid, 0.187g of ferric chloride hexahydrate and 3 mu L of acetic acid in 15mL of N, N-dimethylformamide to obtain a mixture; heating the mixture at 120 ℃ for 4 hours in a condensing reflux mode, and then naturally cooling to room temperature; washing the synthesized product with N, N-dimethylformamide, and drying in a vacuum drying oven at 60 ℃ for 24 hours to obtain Fe-MIL-88 metal organic framework powder;
adding the Fe-MIL-88 metal organic framework powder into deionized water, and performing ultrasonic oscillation until the mixture is uniform to obtain a Fe-MIL-88 metal organic framework solution with the concentration of 0.5 mg/mL;
step 2, adding 10 mu L of aqueous solution of tris (2-carboxyethyl) phosphine with the concentration of 10mmol/L into 250 mu L of capture DNA (dissolved in TE buffer solution) with the concentration of 3 mu mol/L and modified by depurination/pyrimidine sites, and activating for 1 hour at room temperature; meanwhile, 40mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 20mg of N-hydroxysuccinimide were added to 2mL of the Fe-MIL-88 metal-organic framework solution, and stirred for 30 minutes to activate carboxyl groups;
after the activation of the two solutions was completed, they were mixed, shaken at 37 ℃ for 6 hours, and then centrifuged with a 30kDa ultrafiltration tube for 30 minutes, and the resulting precipitate was redispersed in 250. mu.L of TE buffer solution to obtain a dispersion of Fe-MIL-88-captured DNA, which was stored at 4 ℃.
The invention further discloses a method for detecting a target RNA sequence by using the electrochemiluminescence sensor, which comprises the following steps:
step 1, dripping 15 mu L of RNA sample to be detected on the surface of the electrochemiluminescence sensor, incubating for 1 hour at room temperature, and then using 10mmTE buffer solution 8.0 (ol/L, pH), and N2Drying;
step 2, dripping 15 mu L of endonuclease IV solution on the surface of the electrochemiluminescence sensor, carrying out warm bath at 37 ℃ for 125 minutes, then washing with a TE buffer solution with the concentration of 10mmol/L, pH-8.0, and removing the sheared capture DNA fragment and the connected quencher Fe-MIL-88 metal organic framework; with N2Drying to obtain the electrochemiluminescence sensor to be detected;
step 3, measuring an electrochemiluminescence signal of the electrochemiluminescence sensor to be detected through a cyclic voltammetry method to obtain the electrochemiluminescence signal intensity of the RNA sequence to be detected; and then, judging the concentration of the target RNA sequence sample to be detected by utilizing a standard relation curve of the electrochemiluminescence signal intensity and the concentration of the RNA sequence to be detected.
Further, the electrochemiluminescence signal was measured at a signal level of 0.1M K2S2O8The pH of the solution (A) is 7.4, and the solution is carried out in a three-electrode system, wherein the three-electrode system takes an electrochemiluminescence sensor to be detected as a working electrode, a platinum wire as a counter electrode and a silver-silver chloride electrode as a reference electrode.
Further: the potential scanning interval in the cyclic voltammetry is-1.45 to 0V, electrochemiluminescence signals are measured by a photomultiplier under the same high-pressure condition, and the acquisition rate is 50 mV/s.
Further, the standard relation curve is obtained by measuring an electrochemiluminescence signal of an electrochemiluminescence sensor to be detected prepared by using a plurality of concentrations of standard samples of RNA sequences to be detected, and the electrochemiluminescence signal intensity corresponding to each concentration of standard samples of RNA sequences to be detected is obtained; and fitting by taking the logarithm value of the concentration of the standard sample of the RNA sequence to be detected as an abscissa and the intensity of the electrochemiluminescence signal as an ordinate, so as to obtain a standard relation curve of the intensity of the electrochemiluminescence signal and the concentration of the RNA sequence to be detected. Meanwhile, at least 6 point values with different concentrations are taken, and each group of concentrations has at least three groups of parallel data.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention realizes the detection of the target RNA sequence through the electrochemiluminescence sensor, and has the advantages of simple method, wide detection range, high sensitivity and easy operation;
2. according to the invention, by means of a wide ultraviolet absorption interval of the metal organic framework, single particle characteristics and rich exposed groups, the metal organic framework is introduced into detection as a quencher, and signals are amplified by performing depurination/pyrimidine site modification on complementary DNA and circularly shearing hybridized DNA/RNA modified sites by endonuclease IV, so that the sensitivity of the sensor is improved.
3. The invention needs less sample amount for target detection object and has low detection limit.
Drawings
FIG. 1 is a schematic diagram of the preparation process of an electrochemiluminescence sensor for detecting a target RNA sequence according to the present invention.
FIG. 2 is a standard relationship curve of the results of electrochemiluminescence tests performed on the target RNA standard samples of the present invention at concentrations of 300pmol/L, 1.5nmol/L, 3nmol/L, 15nmol/L, 30nmol/L, 150nmol/L, 300nmol/L, 1.5. mu. mol/L, and 3. mu. mol/L, respectively.
FIG. 3 is a Scanning Electron Microscope (SEM) characterization of Fe-MIL-88 metal organic framework nanoparticles of the present invention.
FIG. 4 shows AuNPs of the present invention&g-C3N4And (4) Transmission Electron Microscope (TEM) characterization results of the @ Zr-MOG composite nano material.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. The following is merely exemplary and illustrative of the inventive concept and various modifications, additions and substitutions of similar embodiments may be made to the described embodiments by those skilled in the art without departing from the inventive concept or exceeding the scope of the claims defined thereby.
Example 1
The preparation method of the electrochemiluminescence sensor for detecting RNA sequences in the embodiment is as follows:
1、AuNPs&g-C3N4preparation of a @ Zr-MOG Dispersion:
step 11, g-C3N4Synthesis of @ Zr-MOG Dispersion
Firstly, 3.0mL of g-C with the concentration of 1mg/mL3N4The solution was mixed with 7.0mL of N, N-dimethylformamide and transferred to a reagent bottle; then adding 0.105g of trimesic acid, carrying out ultrasonic treatment for 20 minutes, then adding 0.1165g of anhydrous zirconium chloride, and continuing ultrasonic treatment for 30 minutes to form a uniform solution; heating the obtained homogeneous solution at 120 deg.C for 9 hr to obtain g-C in a reagent bottle3N4@ Zr-MOG cylindrical gel; the obtained g-C3N4Freeze-drying the @ Zr-MOG cylindrical gel at-114 deg.C for 2 days to obtain gel powder;
grinding 20mg of gel powder, dispersing into 20mL of deionized water, and carrying out ultrasonic treatment for 4 hours to obtain g-C3N4@ Zr-MOG dispersion;
step 12, synthesis of AuNPs dispersion
Aqua regia (HNO) for beaker and rotor to be used3: HCl ═ 1: 3) soaking overnight, then washing the beaker and the rotor with deionized water, and drying for later use. In ice bath, adding 0.6mL of 0.1mol/L sodium borohydride solution into 20mL of 250mmol/L chloroauric acid solution, stirring for 10 minutes, reacting the obtained mixture at room temperature for 3 hours to obtain gold nanoparticle AuNPs dispersion, and storing at 4 ℃ for later use;
step 13, AuNPs&g-C3N4Preparation of a @ Zr-MOG Dispersion
To 200. mu.L of deionized water were added 100. mu.L of the AuNPs dispersion obtained in step 2 and 300. mu.L of g-C obtained in step 13N4The @ Zr-MOG dispersion is treated by ultrasonic for 2 hours to obtain AuNPs&g-C3N4@ Zr-MOG dispersion.
2. The capture DNA modified with depurination/pyrimidine sites is linked to the quencher Fe-MIL-88 metal organic framework by amide bonds:
step 21, dissolving 0.126g of 2-aminoterephthalic acid, 0.187g of ferric chloride hexahydrate and 3 μ L of acetic acid in 15mL of N, N-dimethylformamide to obtain a mixture; heating the mixture at 120 ℃ for 4 hours in a condensing reflux mode, and then naturally cooling to room temperature; washing the synthesized product with N, N-dimethylformamide, and drying in a vacuum drying oven at 60 ℃ for 24 hours to obtain Fe-MIL-88 metal organic framework powder;
adding the Fe-MIL-88 metal organic framework powder into deionized water, and performing ultrasonic oscillation until the mixture is uniform to obtain a Fe-MIL-88 metal organic framework solution with the concentration of 0.5 mg/mL;
step 22, adding 10. mu.L of tris (2-carboxyethyl) phosphine aqueous solution with the concentration of 10mmol/L into 250. mu.L of capture DNA (dissolved by TE buffer solution) with the concentration of 3. mu. mol/L and modified by depurination/pyrimidine sites, and activating for 1 hour at room temperature; meanwhile, 40mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 20mg of N-hydroxysuccinimide were added to 2mL of the Fe-MIL-88 metal-organic framework solution, and stirred for 30 minutes to activate carboxyl groups;
after the activation of the two solutions was completed, they were mixed, shaken at 37 ℃ for 6 hours, and then centrifuged with a 30kDa ultrafiltration tube for 30 minutes, and the resulting precipitate was redispersed in 250. mu.L of TE buffer solution to obtain a dispersion of Fe-MIL-88-captured DNA, which was stored at 4 ℃.
3. The glassy carbon electrode was successively coated with Al having a diameter of 0.3 μm on a chamois2O3Powder, Al 0.05 μm in diameter on fleece2O3Polishing the powder until the surface is mirror-like, then washing with deionized water, and then ultrasonically cleaning with deionized water, ethanol and deionized water in sequence to remove Al on the surface2O3And finally with N2Drying;
10 μ L of AuNPs&g-C3N4The @ Zr-MOG dispersion was drop-coated onto the surface of the treated glassy carbon electrode and dried at room temperature.
4. To 15. mu.L of the dispersion of Fe-MIL-88-capture DNA was added 0.6. mu.L of tris (2-carboxyethyl) phosphine aqueous solution at a concentration of 10mmol/L, treated at room temperature for 1 hour to cleave S-S bond, and then added dropwise to the glassCarbon electrode surface and incubation overnight at 4 ℃ followed by rinsing with 10mmol/L, pH ═ 8.0 TE buffer and finally with N2And (5) drying.
5. And (3) dropwise adding 15 mu L of phosphate buffer solution containing 1mmol/L of 6-mercaptohexanol for blocking unreacted active sites onto the surface of a glassy carbon electrode, treating at normal temperature for 1 hour, and then washing with 10mmol/L, pH-8.0 TE buffer solution to remove unreacted 6-mercaptohexanol, so as to obtain the electrochemiluminescence sensor based on the metal organic gel composite material and the metal organic framework, wherein the electrochemiluminescence sensor is used for detecting target RNA.
The method for detecting the target RNA sequence by using the electrochemiluminescence sensor prepared by the embodiment comprises the following steps:
step 1, dropping 15 μ L of RNA sample to be detected on the surface of the electrochemiluminescence sensor, incubating at room temperature for 1 hour, washing with 10mmol/L, pH ═ 8.0 TE buffer solution, and then washing with N2Drying;
step 2, dripping 15 mu L of endonuclease IV solution on the surface of the electrochemiluminescence sensor, carrying out warm bath at 37 ℃ for 125 minutes, then washing with a TE buffer solution with the concentration of 10mmol/L, pH-8.0, and removing the sheared capture DNA fragment and the connected quencher Fe-MIL-88 metal organic framework; with N2Drying to obtain the electrochemiluminescence sensor to be detected;
step 3, measuring an electrochemiluminescence signal of the electrochemiluminescence sensor to be detected by a cyclic voltammetry method to obtain the electrochemiluminescence signal intensity of the RNA sequence to be detected (the electrochemiluminescence signal intensity is output by an MPI-A analyzer (China, Senaemmei analytical instruments, Ltd.), and the signal intensity is recorded after the signal value is stabilized for 300 seconds); and then, judging the concentration of the target RNA sequence sample to be detected by utilizing a standard relation curve of the electrochemiluminescence signal intensity and the concentration of the RNA sequence to be detected.
Specifically, the method comprises the following steps: the electrochemiluminescence signal was measured at a signal level of 0.1M K2S2O8The pH value of 7.4 is carried out in a three-electrode system in a phosphate buffer solution, and the three-electrode system takes an electrochemiluminescence sensor to be detected as a working electrodeThe electrode takes a platinum wire as a counter electrode and a silver-silver chloride electrode as a reference electrode; the potential scanning interval in the cyclic voltammetry is-1.45 to 0V, the electrochemiluminescence signals are measured by a photomultiplier under the same high-pressure condition, and the acquisition rate is 50 mV/s.
The standard relationship curve in this embodiment is obtained by the following method: preparing a plurality of RNA sequence standard samples to be detected with a certain concentration, and respectively measuring electrochemiluminescence signals of the samples according to the detection method to obtain the electrochemiluminescence signal intensity corresponding to the RNA sequence standard samples to be detected with the certain concentration; and fitting by taking the logarithm value of the concentration of the standard sample of the RNA sequence to be detected as an abscissa and the intensity of the electrochemiluminescence signal as an ordinate, so as to obtain a standard relation curve of the intensity of the electrochemiluminescence signal and the concentration of the RNA sequence to be detected.
Specifically, in this embodiment, the concentration of the standard sample of the RNA sequence to be detected is 300pmol/L to 3 μmol/L, and each group of concentrations has at least three groups of parallel data. As shown in FIG. 2, the standard relationship curve obtained in this example is IECL＝1153.46logCRNA+14205.05. The detection shows that when the concentration of the RNA sample is in the range of 300pmol/L to 3 mu mol/L, the electrochemiluminescence intensity increases along with the increase of the concentration of the RNA sequence, and the electrochemiluminescence intensity is in a linear relation with the concentration, and the detection limit reaches 100 pmol/L.
In order to verify the accuracy of the electrochemiluminescence sensor in the embodiment in detection in actual samples, a recovery rate test was performed. 10-fold dilution of human serum was performed with TE buffer, and target RNA concentrations of 3nmol/L, 30nmol/L, and 300nmol/L were added thereto, respectively. The concentrations of each sample were calculated as 2.76nmol/L, 3.04nmol/L, and 3.11nmol/L using the above method, with relative standard deviations of 5.8%, 1.0%, and 1.5%, respectively. Recovery rate experiments indicate that the sensor will have good application in clinical measurements.
TABLE 1 nucleotide sequences of DNA and RNA used in example 1
Figure BDA0002403166320000071
X represents a modified depurination/pyrimidine site.

Claims (9)

1. An electrochemiluminescence sensor based on a metal organic gel composite material and a metal organic framework is characterized in that: the electrogenerated chemiluminescence sensor is characterized in that AuNPs are fixed on the surface of a glassy carbon electrode&g-C3N4@ Zr-MOG composite nanomaterial, in which AuNPs&g-C3N4Fixing capture DNA modified by depurination/pyrimidine sites on the surface of the @ Zr-MOG composite nano material through a gold-sulfur bond; the other end of the capture DNA modified by the depurination/pyrimidine site is connected with a quencher Fe-MIL-88 metal organic framework through an amido bond;
the sequence of the capture DNA is complementary with the base of the sequence of the RNA to be detected.
2. The electrochemiluminescence sensor according to claim 1, wherein: the AuNPs&g-C3N4The @ Zr-MOG composite nano material and the quencher Fe-MIL-88 metal organic framework are linked through capture DNA modified by depurination/pyrimidine sites, so that the electrochemiluminescence signal is quenched;
when an RNA sequence to be detected is dripped on the electrochemiluminescence sensor, capturing DNA and the base of the RNA sequence to be detected are complementarily paired, and then a specific cutting site of endonuclease IV is added to remove the cut capturing DNA segment and a quencher Fe-MIL-88 metal organic framework connected with the cut capturing DNA segment, so that the electrochemiluminescence signal is gradually recovered, and the electrochemiluminescence sensor to be detected is obtained;
and testing the electrochemiluminescence signal of the electrochemiluminescence sensor to be tested to obtain the electrochemiluminescence signal intensity of the RNA sequence to be tested, and judging the concentration of the RNA sequence sample to be tested by utilizing a standard relation curve of the electrochemiluminescence signal intensity and the concentration of the RNA sequence to be tested.
3. The electrochemiluminescence sensor according to claim 1, wherein: the AuNPs&g-C3N4The @ Zr-MOG composite nano material is doped with g-C3N4Au nano-particles are uniformly loaded on the Zr metal organic gel through electrostatic adsorption.
4. A method for preparing an electrochemiluminescence sensor according to any of claims 1 to 3, comprising the steps of:
step 1, sequentially putting glassy carbon electrodes on chamois and using Al with the diameter of 0.3 mu m2O3Powder, Al 0.05 μm in diameter on fleece2O3Polishing the powder until the surface is mirror-like, then washing with deionized water, and then ultrasonically cleaning with deionized water, ethanol and deionized water in sequence to remove Al on the surface2O3And finally with N2Drying;
10 μ L of AuNPs&g-C3N4The @ Zr-MOG dispersed liquid is dripped on the surface of the glassy carbon electrode after treatment and is dried at room temperature;
step 2, connecting the capture DNA modified by the depurination/pyrimidine site with a quencher Fe-MIL-88 metal organic framework through an amido bond to obtain a dispersion liquid of the Fe-MIL-88 capture DNA;
to 15. mu.L of the dispersion of Fe-MIL-88-captured DNA was added 0.6. mu.L of tris (2-carboxyethyl) phosphine aqueous solution at a concentration of 10mmol/L, treated at room temperature for 1 hour to cleave S-S bonds, then dropped onto the surface of a glassy carbon electrode, and incubated overnight at 4 ℃, washed with 10mmol/L, pH ═ 8.0 TE buffer solution, and finally N was added2Drying;
and 3, dropwise adding 15 mu L of phosphate buffer solution containing 1mmol/L of 6-mercaptohexanol for blocking unreacted active sites onto the surface of the glassy carbon electrode, treating at normal temperature for 1 hour, and then washing with 10mmol/L, pH-8.0 TE buffer solution to remove unreacted 6-mercaptohexanol, so as to obtain the electrochemiluminescence sensor based on the metal organic gel composite material and the metal organic framework, wherein the electrochemiluminescence sensor is used for detecting target RNA.
5. The method of claim 4, wherein: the AuNPs&g-C3N4The @ Zr-MOG dispersion was prepared as follows:
step 1, g-C3N4Synthesis of @ Zr-MOG Dispersion
Firstly, 3.0mL of g-C with the concentration of 1mg/mL3N4The solution was mixed with 7.0mL of N, N-dimethylformamide and transferred to a reagent bottle; then adding 0.105g of trimesic acid, carrying out ultrasonic treatment for 20 minutes, then adding 0.1165g of anhydrous zirconium chloride, and continuing ultrasonic treatment for 30 minutes to form a uniform solution; heating the obtained homogeneous solution at 120 deg.C for 9 hr to obtain g-C in a reagent bottle3N4@ Zr-MOG cylindrical gel; the obtained g-C3N4Freeze-drying the @ Zr-MOG cylindrical gel at-114 deg.C for 2 days to obtain gel powder;
grinding 20mg of gel powder, dispersing into 20mL of deionized water, and carrying out ultrasonic treatment for 4 hours to obtain g-C3N4@ Zr-MOG dispersion;
step 2, synthesis of AuNPs dispersion liquid
In ice bath, adding 0.6mL of 0.1mol/L sodium borohydride solution into 20mL of 250mmol/L chloroauric acid solution, stirring for 10 minutes, reacting the obtained mixture at room temperature for 3 hours to obtain gold nanoparticle AuNPs dispersion, and storing at 4 ℃ for later use;
step 3, AuNPs&g-C3N4Preparation of a @ Zr-MOG Dispersion
To 200. mu.L of deionized water were added 100. mu.L of the AuNPs dispersion obtained in step 2 and 300. mu.L of g-C obtained in step 13N4The @ Zr-MOG dispersion is treated by ultrasonic for 2 hours to obtain AuNPs&g-C3N4@ Zr-MOG dispersion.
6. The method according to claim 4, wherein the capture DNA modified with depurination/pyrimidine site is linked to a quencher Fe-MIL-88 metal-organic framework via an amide bond by:
step 1, dissolving 0.126g of 2-aminoterephthalic acid, 0.187g of ferric chloride hexahydrate and 3 mu L of acetic acid in 15mLN, N-dimethylformamide to obtain a mixture; heating the mixture at 120 ℃ for 4 hours in a condensing reflux mode, and then naturally cooling to room temperature; washing the synthesized product with N, N-dimethylformamide, and drying in a vacuum drying oven at 60 ℃ for 24 hours to obtain Fe-MIL-88 metal organic framework powder;
adding the Fe-MIL-88 metal organic framework powder into deionized water, and performing ultrasonic oscillation until the mixture is uniform to obtain a Fe-MIL-88 metal organic framework solution with the concentration of 0.5 mg/mL;
step 2, adding 10 mu L of tris (2-carboxyethyl) phosphine aqueous solution with the concentration of 10mmol/L into 250 mu L of capture DNA modified by depurination/pyrimidine sites with the concentration of 3 mu mol/L, and activating for 1 hour at room temperature; meanwhile, 40mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 20mg of N-hydroxysuccinimide were added to 2mL of the Fe-MIL-88 metal-organic framework solution, and stirred for 30 minutes to activate carboxyl groups;
after the activation of the two solutions was completed, they were mixed, shaken at 37 ℃ for 6 hours, and then centrifuged with a 30kDa ultrafiltration tube for 30 minutes, and the resulting precipitate was redispersed in 250. mu.L of TE buffer solution to obtain a dispersion of Fe-MIL-88-captured DNA, which was stored at 4 ℃.
7. A method for detecting a target RNA sequence using the electrochemiluminescence sensor as set forth in any one of claims 1 to 3, comprising the steps of:
step 1, dropping 15 μ L of RNA sample to be detected on the surface of the electrochemiluminescence sensor, incubating at room temperature for 1 hour, washing with TE buffer solution of 10mmol/L, pH ═ 8.0, and then washing with N2Drying;
step 2, dripping 15 mu L of endonuclease IV solution on the surface of the electrochemiluminescence sensor, carrying out warm bath at 37 ℃ for 125 minutes, then washing with a TE buffer solution with the concentration of 10mmol/L, pH-8.0, and removing the sheared capture DNA fragment and the connected quencher Fe-MIL-88 metal organic framework; with N2Drying to obtain the electrochemiluminescence sensor to be detected;
step 3, measuring an electrochemiluminescence signal of the electrochemiluminescence sensor to be detected through a cyclic voltammetry method to obtain the electrochemiluminescence signal intensity of the RNA sequence to be detected; and then, judging the concentration of the target RNA sequence sample to be detected by utilizing a standard relation curve of the electrochemiluminescence signal intensity and the concentration of the RNA sequence to be detected.
8. The method of claim 7, wherein: the electrochemiluminescence signal is measured at a signal level of 0.1M K2S2O8The pH of the solution (A) is 7.4, and the solution is carried out in a three-electrode system, wherein the three-electrode system takes an electrochemiluminescence sensor to be detected as a working electrode, a platinum wire as a counter electrode and a silver-silver chloride electrode as a reference electrode.
9. The method of claim 7, wherein: the standard relation curve is obtained by measuring an electrochemiluminescence signal of an electrochemiluminescence sensor to be detected prepared by using RNA sequence standard samples to be detected with a plurality of concentrations to obtain the electrochemiluminescence signal intensity corresponding to each RNA sequence standard sample to be detected with each concentration; and fitting by taking the logarithm value of the concentration of the standard sample of the RNA sequence to be detected as an abscissa and the intensity of the electrochemiluminescence signal as an ordinate, so as to obtain a standard relation curve of the intensity of the electrochemiluminescence signal and the concentration of the RNA sequence to be detected.
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