CN111020006B - Electrochemical luminescence sensor system for measuring adenosine triphosphate, and preparation method and application thereof - Google Patents

Electrochemical luminescence sensor system for measuring adenosine triphosphate, and preparation method and application thereof Download PDF

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CN111020006B
CN111020006B CN201911156060.1A CN201911156060A CN111020006B CN 111020006 B CN111020006 B CN 111020006B CN 201911156060 A CN201911156060 A CN 201911156060A CN 111020006 B CN111020006 B CN 111020006B
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沈艳飞
宁振强
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Abstract

The invention discloses an electrochemiluminescence sensor system for measuring adenosine triphosphate, a preparation method and application thereof, wherein the system consists of a magnetic probe for amplifying an adenosine triphosphate concentration signal and an electrochemiluminescence sensor for measuring Trigger DNA; the magnetic probe is made of Fe 3 O 4 The nano-particles, the gold nano-particles, the DNA substrate and the aptazyme are compounded in sequence; electrochemical luminescence sensor connects amino modified Capture DNA to modified RuSiO through glutaraldehyde crosslinking 2 The working electrode surface of CS. In the invention, adenosine triphosphate firstly participates in a constant-temperature amplification reaction guided by a magnetic probe, and generates a large amount of intermediate Trigger DNA; then, the electrochemical luminescence sensor of the invention is used for carrying out quantitative detection on the Trigger DNA of the intermediate, and finally, the aim of indirectly and quantitatively detecting the adenosine triphosphate is achieved.

Description

Electrochemical luminescence sensor system for measuring adenosine triphosphate, and preparation method and application thereof
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to an electrochemiluminescence sensor system for detecting adenosine triphosphate, and a preparation method and application thereof.
Background
Adenosine Triphosphate (ATP) is a small molecule high-energy phosphate compound, and is also a main energy source in organic life bodies, so that energy supply of various vital activities of cells is guaranteed. As an "energy currency", it participates in many important physiological processes in living beings, such as: gene synthesis, nutrient metabolism, drug delivery, and regulation of immune and neural-mediated biological activities. Related researches show that the change of ATP content as an indicator of cell viability and cell damage of organic life bodies is closely related to the occurrence of diseases such as cardiovascular diseases, parkinson's disease, alzheimer's disease and the like. In addition, in the field of food safety, quantitative detection of ATP is also used for detection of food-borne pathogenic microorganisms. Therefore, the ATP quantitative detection method with high sensitivity and high specificity is established, and has intuitive and important effects on the fields of life science research, clinical diagnosis, food safety, environmental analysis and the like.
Various detection methods have been developed for the quantitative detection of ATP, such as: capillary electrophoresis, high performance liquid chromatography, mass spectrometry, chemiluminescence, etc. The detection method has the advantages of accuracy, high efficiency and the like, but also has the defects of complicated and time-consuming sample processing steps, low sensitivity, huge equipment, high cost and the like, and limits the wide application of the detection method in different fields to a certain extent. Therefore, in order to meet the research and application needs in various fields, it is urgently needed to develop a simple, rapid and high-sensitivity ATP quantitative detection method.
Electrochemiluminescence (ECL) is an electrochemically driven chemiluminescence phenomenon that has superior performance compared to other optical detection methods by intelligently integrating electrochemical and chemiluminescence techniques. The electrochemiluminescence does not need an additional light source, so that experimental equipment is simplified, background interference of magazines and scattering light sources is avoided, and the electrochemiluminescence has higher sensitivity. In addition, the electrochemiluminescence has the advantages of high specificity, simplicity in operation, good reproducibility and the like, attracts the attention of a plurality of researchers, is widely applied to the fields of food and medicine analysis, environmental monitoring and the like, and is one of the most potential platforms for realizing the quantitative detection of the advantageous ATP. In order to develop a sensor meeting the requirements of the current ATP quantitative detection, the invention integrates a DNA signal amplification strategy and the excellent luminescence effect of the nano material, thereby realizing a multiple signal amplification system and being successfully applied to the ATP quantitative detection.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects of complexity, time consumption, low sensitivity, huge equipment, high cost and the like in the prior art, the invention provides an electrochemical luminescence sensor system for measuring adenosine triphosphate.
Another object of the present invention is to provide a method for preparing the ATP electrochemiluminescence sensor system.
It is another object of the invention to provide the use of such an adenosine triphosphate electrochemiluminescence sensor system. The invention provides a method for quantitatively detecting adenosine triphosphate in human serum by using the sensor system, and the detection method has the advantages of good specificity, high sensitivity, wide linear range and low cost.
The technical scheme is as follows: in order to achieve the above object, the present invention provides an electrochemiluminescence sensor system for measuring adenosine triphosphate, which comprises a magnetic probe Au @ Fe3O4-substrate-aptamer for amplifying adenosine triphosphate concentration signal and an electrochemiluminescence sensor for measuring Trigger DNA; the magnetic probe Au @ Fe 3 O 4 -substrate-aptamer from Fe 3 O 4 The nano-particles, the gold nano-particles, the DNA substrate and the aptazyme are compounded in sequence; the electrochemiluminescence sensor for measuring Trigger DNA connects amino modified Capture DNA to modified RuSiO through glutaraldehyde crosslinking 2 -the working electrode surface of CS.
In the invention, adenosine triphosphate firstly participates in a constant-temperature amplification reaction guided by a magnetic probe, and generates a large amount of intermediate Trigger DNA; then, the electrochemical luminescence sensor of the invention is used for carrying out quantitative detection on the Trigger DNA of the intermediate, and finally, the aim of indirectly and quantitatively detecting the adenosine triphosphate is achieved.
Wherein the DNA substrate sequence is SH-AAAAAAAATTCACCAACTAATrAGCTACGATGACTCACCTAGGAG; the aptazyme sequence is 'TCATCGTAGAGCGATCTAGGGGGAGTATTGCGGAGGATGCAGCACCCATGTTAGTTGGTAA'; the Trigger DNA sequence is 'GCTACGATGACTCACCTAGGAG'; the amino modified Capture DNA sequence is' NH 2 -ctcttagggtgagtcatcgagcctcctcctggtattgctacgatgactc a "; the sequences are respectively shown by SEQ ID NO. 1-4.
The intermediate Trigger DNA is a DNA fragment generated by the enzyme digestion of DNA substrate by aptazyme, and has the function of using a magnetic probe Au @ Fe 3 O 4 The substrate-aptamer mediated signal amplification strategy is organically linked to the sensor detection: after ATP is combined with the magnetic probe, the enzyme digestion activity of the aptazyme is started and specifically cuts a DNA substrate chain, and Trigger DNA is finally generated; because the concentration of the product Trigger DNA is in a direct proportion relation with the ATP concentration, the sensor can realize the quantitative detection of ATP by detecting the Trigger DNA.
The invention relates to a preparation method of an electrochemiluminescence sensor system for measuring adenosine triphosphate, which comprises the following steps:
magnetic probe Au @ Fe 3 O 4 Preparation of-substrate-aptamer enzyme
(1) Weighing FeCl 3 And dissolving trisodium citrate in ethylene glycol, adding sodium acetate, adding the stirred mixed solution into a reaction kettle, and cleaning the precipitate after reaction to obtain Fe 3 O 4 Nanoparticles;
(2) To HAuCl 4 Adding a trisodium citrate aqueous solution into the aqueous solution, and continuously boiling to obtain Au nanoparticles;
(3) Using APTES on Fe 3 O 4 Performing amination modification on the nanoparticles, mixing the product with Au nanoparticles, performing magnetic separation, and cleaning to obtain Au @ Fe 3 O 4 Nanoparticles;
(4)Au@Fe 3 O 4 mixing the nano particles and DNA substrate, carrying out magnetic separation after reaction, and then washing with water to obtain Au @ Fe3O4-substrate solution; then mixed with aptazymeSynthesizing and reacting overnight to obtain an Au @ Fe3O4-substrate-aptazyme probe;
preferably, the aforementioned Au @ Fe 3 O 4 The preparation method of the substrate-aptamer probe comprises the following steps:
(1) Weighing FeCl 3 And trisodium citrate are dissolved in ethylene glycol, sodium acetate is added, the mixture is added into a reaction kettle after magnetic stirring is carried out for 10-60 min, and the mixture reacts for 6-16 h at 200 ℃; the precipitate is washed by ethanol and water in sequence after magnetic separation to obtain Fe 3 O 4 Nanoparticles;
(2) 0.01% (mass fraction) HAuCl to boiling 4 Adding 0.1 percent (mass fraction) trisodium citrate aqueous solution into the aqueous solution, and continuously boiling for 10-30 min to obtain Au nano particles;
(3) Using APTES on Fe 3 O 4 Performing amination modification on the nano particles, mixing the product with Au nano particles, performing magnetic separation, and then washing with water to obtain Au @ Fe 3 O 4 Nanoparticles;
(4)Au@Fe 3 O 4 mixing the nano particles with DNA substrate, reacting at 37 ℃ for 12-36 h, magnetically separating, and washing with water to obtain Au @ Fe 3 O 4 -a substrate solution; then mixing with aptazyme, reacting at 37 ℃ overnight to obtain Au @ Fe 3 O 4 -a substrate-aptazyme probe;
FeCl in the step (1) 3 The weight ratio of the trisodium citrate to the sodium acetate is as follows: 0.65:0.2:1.2.
The HAuCl in the step (2) 4 And the volume ratio of the trisodium citrate aqueous solution is 50.
The final concentration of the APTES in the step (3) is 1-10% (volume fraction) of the total reaction liquid volume, and Fe 3 O 4 The concentration of the nano particles is 0.5-5 mg/mL.
Step (4) the Au @ Fe 3 O 4 The concentration of the nano particles is 0.5-5 mg/mL, the concentration of DNA substrate is 1-10 mu M, and the concentration of the aptazyme is 1-10 mu M.
More preferably:
(1) 0.65g FeCl was weighed 3 And 0.2g trisodium citrate dissolved in 20mL of ethylene glycol, added1.2g of sodium acetate, magnetically stirring the mixture for 30min, adding the mixture into a reaction kettle, and reacting for 10h at 200 ℃; the precipitate is washed by ethanol and water in sequence after magnetic separation to obtain Fe 3 O 4 Nanoparticles;
(2) 50mL 0.01% to boiling of HAuCl 4 Adding 1mL of 0.1% trisodium citrate aqueous solution into the aqueous solution, and continuously boiling for 15min to obtain Au nanoparticles;
(3) Adding 1% of APTES to 1mg/mL Fe in the total reaction volume 3 O 4 Subjecting the nanoparticles to amination modification, mixing the product with Au nanoparticles, performing magnetic separation, and washing with water to obtain Au @ Fe 3 O 4 Nanoparticles.
(4)1mg/mL Au@Fe 3 O 4 Mixing the nanoparticle water solution with 1 μ M DNA substrate, reacting at 37 deg.C for 24h, magnetically separating, and washing with water to obtain Au @ Fe 3 O 4 -a substrate solution; then mixed with 1 μ M aptazyme, reacted at 37 ℃ overnight to obtain magnetic probe Au @ Fe 3 O 4 Substrate-aptazyme solution.
The invention discloses a preparation method of an electrochemiluminescence sensor system for measuring adenosine triphosphate, and RuSiO 2 -CS is from RuSiO 2 Mixing the aqueous solution and the CS solution, and fully and uniformly mixing to obtain the compound; the RuSiO 2 To add Ru (bpy) to a mixture of Triton X-100, cyclohexane and n-hexanol 3 Cl 2 Mixing the aqueous solution evenly, adding TEOS and ammonia water, and stirring for reaction; adding acetone, centrifuging, cleaning the precipitate to obtain RuSiO 2 。
The RuSiO 2 The preparation method comprises the following steps:
adding Ru (bpy) into a mixture of Triton X-100, cyclohexane and hexanol 3 Cl 2 Mixing the aqueous solution evenly, adding TEOS and ammonia water, stirring and reacting for 12-36 h; adding acetone, centrifuging, sequentially cleaning precipitate with ethanol and water to obtain RuSiO 2 。
Preferably, 1-3 mL of Triton X-100, 5-10 mL of cyclohexane is measured, 1-3 mL of hexanol is added into a reaction vessel, after thorough mixing, 250-500. Mu.L of 5-100mM Ru (bpy) is added into the mixed solution 3 Cl 2 Mixing the aqueous solution evenly, adding 50-200 mu L TEOS and 30-200 mu L ammonia water, and stirring for reaction for 12-36 h; adding 1-10 mL of acetone, centrifuging, washing the precipitate with ethanol and water in sequence, and resuspending the product with ethanol to obtain 1-8 mg/mL RuSiO 2 And (3) solution.
The RuSiO 2 The CS solution was prepared as follows:
adding CS into an acetic acid aqueous solution and carrying out ultrasonic dissolution to obtain a CS solution; taking equal volume of RuSiO 2 Mixing the solution with the CS solution, and performing ultrasonic treatment for 10-50 min to obtain RuSiO 2 -a CS solution.
Preferably, 0.1-2 mg of CS is added into 0.1-2 mL of acetic acid aqueous solution with volume fraction of 0.5% -3%, and the CS solution is obtained after ultrasonic dispersion for 5-30 min; taking 0.1-2mL 1-8 mg/mL RuSiO 2 Adding the solution into 0.1-2 mL of CS solution, and carrying out ultrasonic treatment for 10-60 min to obtain uniformly dispersed RuSiO 2 -a CS solution.
The preparation process of the electrochemical luminescence sensor is as follows:
(1) Polishing the working electrode, and then ultrasonically cleaning;
(2) Taking RuSiO 2 Dripping a CS solution on the surface of the working electrode, standing and drying at room temperature, and cleaning and airing;
(3) Dripping glutaraldehyde aqueous solution on the surface of the electrode, reacting at room temperature, and then cleaning and drying; dropwise adding the Capture DNA to the surface of an electrode, and cleaning and airing after reaction;
(4) Dropwise adding Blocker to the surface of the electrode, reacting at room temperature, and then cleaning and drying;
preferably, the working electrode is a glassy carbon electrode.
Preferably, the preparation method of the electrochemical luminescence sensor comprises the following steps:
(1) The working electrode was successively treated with 0.3 μm and 0.05 μm Al 2 O 3 Polishing the powder, and then ultrasonically cleaning the powder for 2 to 10min by using water, ethanol and water in sequence;
(2) Taking 2-20 mu L of 1-8 mg/mL RuSiO 2 Dropping CS solution on the surface of the working electrode, standing and drying at room temperature, and adding PBS solutionCleaning and drying;
(3) Dripping glutaraldehyde aqueous solution with the mass fraction of 0.5-5% onto the surface of the electrode, reacting at room temperature for 1-3 h, cleaning with PBS solution, and drying in the air; dripping 2-20 mu L of 1-10 mu M Capture DNA on the surface of an electrode, reacting for 1-3 h at 37 ℃, cleaning with a PBS solution, and drying in the air;
(4) Dripping 2-20 mu L of 1-10 mu M of Blocker on the surface of the electrode, reacting for 1-3 h at room temperature, cleaning with a PBS solution, and drying in the air;
more preferably still, the first and second liquid crystal compositions are,
(1) The working electrode was successively treated with 0.3 μm and 0.05 μm Al 2 O 3 Polishing the powder, and then ultrasonically cleaning for 4min by using water, ethanol and water in sequence;
(2) Taking 10 mu L of 2mg/mL RuSiO 2 Dripping a CS solution on the surface of the working electrode, standing and drying at room temperature, washing with a PBS solution, and drying in the air;
(3) Dripping 2.5 mass percent of glutaraldehyde aqueous solution on the surface of the electrode, reacting for 2 hours at room temperature, cleaning with PBS solution, and drying in the air; dripping 10 mu L of 4 mu M Capture DNA on the surface of an electrode, reacting for 2h at 37 ℃, washing with a PBS solution, and drying in the air;
(4) Dripping 2-20 mu L of 1-10 mu M of Blocker on the surface of the electrode, reacting for 1-3 h at room temperature, cleaning with a PBS solution, and drying in the air;
wherein the Blocker sequence is NH 2 -TTTTTTTT。
The invention relates to an application of an electrochemiluminescence sensor system for measuring adenosine triphosphate in the preparation of a tool or a reagent for quantitatively detecting the adenosine triphosphate.
The quantitative detection of the adenosine triphosphate comprises the following detection operation steps:
(1) Adenosine triphosphate solution and magnetic probe Au @ Fe 3 O 4 -substrate-aptamer mixing and reaction for a period of time, separating bound complexes of adenosine triphosphate and magnetic probes using magnetic adsorption;
(2) Resuspending the compound by using enzyme digestion buffer solution, activating enzyme digestion reaction at a specific temperature (37 ℃) to generate a large amount of Trigger DNA, and removing the magnetic probe by magnetic adsorption to obtain supernatant solution containing the Trigger DNA;
(3) And mixing the solution with hairpin-Fc in the same volume, dropwise adding the mixture on the surface of the electrochemical luminescence sensor, reacting for a period of time, testing an electrochemical luminescence signal of the sensor, and calculating the corresponding ATP concentration according to the attenuation value of the electrochemical luminescence signal.
Specifically, the sensor was placed in a 0.01M PBS solution containing 25mM triethylamine for CV testing at a potential range of 0 to 1.3V and a rate of 0.1V/s. ECL luminescence signals were recorded simultaneously with a photomultiplier high voltage (PMT) of 800V. And calculating the attenuation value of the ECL signal, and calculating the concentration of the corresponding adenosine triphosphate according to the standard curve.
Wherein the hairpin-Fc sequence is TCGTAGCAATACCAGGAGGCTACGATGACTCACTCCTGGTAT-Fc, and the sequence is shown by SEQ ID NO. 5.
As a preference, the first and second liquid crystal compositions are,
(1) Mixing ATP solution to be detected with Au @ Fe 3 O 4 Mixing a substrate-aptamer probe, reacting at 37 ℃ for 0.5-3 h, and performing magnetic separation;
(2) With digestion buffer (containing 100mM NaCl and 20mM MgCl) 2 20mM HEPES buffer solution) resuspending the adenosine triphosphate-adsorbing magnetic probe in the step (1), reacting for 2h at 37 ℃, and magnetically separating;
(3) Mixing the supernatant with hairpin-Fc solution in equal volume, dripping 5-50 mu L of the mixed solution on the surface of an electrode, reacting for 0.5-3 h at 37 ℃, washing with PBS solution, and drying in the air;
(4) And testing the ECL luminescence signal of the sensor.
More preferably, the amount of the organic solvent is,
(1) Taking 100 μ L ATP solution to be detected, and mixing with 100 μ L Au @ Fe 3 O 4 Mixing a substrate-aptamer enzyme probe, reacting for 2 hours at 37 ℃, and performing magnetic separation;
(2) mu.L of digestion buffer (containing 100mM NaCl and 20mM MgCl) 2 20mM HEPES buffer) resuspending the adenosine triphosphate-adsorbing magnetic probe in step (1), reacting for 2h at 37 ℃, and magnetically separating;
(3) Mixing the supernatant with 2 mu M hairpin-Fc solution in equal volume, dripping 10 mu L of the mixture on the surface of an electrode, reacting for 2h at 37 ℃, washing with PBS solution, and drying in the air;
(4) And testing the ECL luminescence signal of the sensor.
The pH of PBS used for washing in the present invention was 7.4.
The invention relates to an electrochemiluminescence sensor system for measuring adenosine triphosphate, which has the detection principle that: the magnetic probe Au @ Fe3O4-substrate-aptazyme is used for specific capture of adenosine triphosphate molecules, and simultaneously starts the isothermal enzyme amplification reaction of the aptazyme to generate a large amount of Trigger DNA; under the catalytic action of Trigger DNA, hairpin-Fc is complementarily combined with Capture DNA modified on RuSiO2-CS on the surface of the sensor, so that a large amount of hairpin-Fc is fixed on the surface of an electrode; since Fc can inhibit RuSiO2-CS electrochemical luminescence signal dose-dependently, the concentration of adenosine triphosphate and electrochemical luminescence signal attenuation value are linear relation. Based on the principle, the electrochemical luminescence sensor system realizes the quantitative detection of adenosine triphosphate.
Abbreviations for technical terms in the present invention are as follows:
adenosine triphosphate, ATP; silica microsphere doped with ruthenium terpyridyl chloride RuSiO 2 (ii) a CS as chitosan; si (OC) 2 H 5 ) 4 :TEOS；H 2 NCH 2 CH 2 CH 2 Si(OC 2 H 5 ) 3 APTES; ferrocene: fc.
The nucleic acid sequences of Trigger DNA, capture DNA, DNA substrate, aptazyme, blocker, hairpin-Fc, etc. involved in the present invention were synthesized by Shanghai Biotechnology engineering (Shanghai) Ltd. Other starting reagents in the present invention are commercially available. ATP, TEOS, APTES, HAuCl 4 Chitosan was purchased from Sigma company, usa, and the rest of chemical reagents were domestic analytical pure and purchased from chemical reagents limited of shanghai pharmaceutical group.
During the physiological activities of a living body, adenosine triphosphate is often in a matrix with complex components, which can cause the generation of false positive results to some extent in the actual detection process. The present invention attempts to enhance the sensitivity of the assay by amplifying signals using isothermal nucleic acid amplification, which is a way to effectively reduce the matrix effect. Meanwhile, the magnetic separation technology is an effective means which can purify the target analyte from a complex matrix and effectively eliminate the effect of the sample matrix at present. Therefore, the invention tries to effectively combine two nucleic acid constant-temperature amplification methods with a magnetic separation technology and design and prepare an electrochemical luminescence sensor system for measuring the adenosine triphosphate by utilizing the high-sensitivity characteristic of an electrochemical luminescence platform.
The invention designs a novel sensor system with a double signal amplification strategy aiming at the particularity of a target object, wherein the sensor is formed by connecting aptamer Enzyme and Enzyme-Free constant-temperature signal amplification technology based on catalytic hairpin self-assembly in series, and then is cut in from the signal amplification angle of a luminescent material to synthesize RuSiO of silicon dioxide-coated terpyridine ruthenium chloride molecules 2 The nano-particles are combined with the signal amplification strategy to obtain a novel electrochemical luminescence sensor with excellent performance and multiple signal amplification capability. In the detection process, the aptamer enzyme is specifically combined with adenosine triphosphate and cuts a substrate probe in a sensor system to obtain a large amount of Trigger DNA, the Trigger DNA triggers a catalytic Hairpin self-assembly reaction on the surface of an electrode to generate Capture DNA-Hairpin-Fc double-stranded DNA, and then the detection of the luminescence signal value of the sensor is completed on an electrochemical luminescence testing instrument. Due to Fc, ruSiO can be effectively quenched 2 The electrochemical luminescence signal of (2), therefore, the ultra-sensitive detection of the adenosine triphosphate can be realized through the signal attenuation values before and after the test. In addition, the sensor system successfully realizes the quantitative detection of the adenosine triphosphate in the human serum, and the detection method has the advantages of good specificity, high sensitivity, wide linear range and low cost.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) The electrochemical luminescence sensor system is applied to detecting adenosine triphosphate in serum and bacteria cells, does not need complex treatment on samples in conventional detection methods, and also avoids the problems of high cost, complex operation and the like of the conventional detection methods such as HPLC (high performance liquid chromatography).
(2) Compared with the existing commercial adenosine triphosphate detection kit and the reported electrochemical luminescence sensor, the electrochemical luminescence sensor system has higher sensitivity.
(3) Compared with other electrochemical luminescence sensors, the electrochemical luminescence sensor system disclosed by the invention applies a multiple signal amplification strategy and has the advantages of high sensitivity, high specificity and the like; meanwhile, the invention effectively reduces false positive results in the actual detection process by utilizing the magnetic separation technology.
(4) The invention successfully prepares the high-efficiency luminescent material RuSiO 2 And the method is successfully combined with signal amplification strategies such as aptamer enzyme and catalytic hairpin self-assembly, and has certain guiding significance, theoretical value and practical value for development of multiple signal amplification strategies in the field of biosensing.
(5) The invention designs an electrochemiluminescence sensor system based on a multiple signal amplification strategy aiming at the ATP particularity of a target analyte, the sensor system successfully realizes the quantitative detection of adenosine triphosphate in human serum, and has the advantages of good specificity, high sensitivity, wide linear range, low cost, convenient use and the like.
Drawings
FIG. 1 is a schematic diagram of the design and fabrication of an electrochemiluminescence sensor according to the present invention;
FIG. 2 is a cyclic voltammogram of an electrochemiluminescence sensor of the present invention;
FIG. 3 is a schematic diagram showing the relationship between the incubation time of the ECL system according to the present invention for the enzyme-cleaved product and the ECL intensity;
FIG. 4 is a graph of the linear relationship between ECL intensity and log of antigen concentration for an electrochemiluminescence sensor system of the present invention;
FIG. 5 is a diagram illustrating an alternative embodiment of an electrochemiluminescence sensor system according to the present invention;
FIG. 6 is a schematic diagram of the feasibility of the electrochemiluminescence sensor system of the present invention for quantitative determination of ATP in human serum.
Detailed Description
The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims.
Example 1
Au@Fe 3 O 4 Preparation of-substrate-aptamer Probe
(1)Fe 3 O 4 Preparation of nanoparticles
0.65g FeCl was weighed 3 Adding 0.2g of trisodium citrate into a beaker containing 20mL of ethylene glycol, fully stirring until the trisodium citrate is completely dissolved, adding 1.2g of sodium acetate into the beaker, carrying out magnetic stirring for 30min, adding the mixed solution into a reaction kettle, and reacting for 10h at 200 ℃; magnetic separating the precipitate, washing with ethanol and water for three times to obtain Fe 3 O 4 Nanoparticles.
(2) Preparation of Au nanoparticles
Prepared 50mL of HAuCl with a mass fraction of 0.01% 4 Adding the aqueous solution into a round-bottom flask, heating to boil, adding 1mL of trisodium citrate aqueous solution with the mass fraction of 0.1%, continuously heating to boil for 15min, and cooling the sample to room temperature to obtain the Au nanoparticle solution.
(3)Au@Fe 3 O 4 Nanoparticle preparation
10mg of Fe are weighed 3 O 4 Adding the nano particles into 10mL of ethanol solution, fully stirring to fully disperse the nano particles, adding 100uL of APTES solution into the solution, reacting at room temperature for 10h to obtain the amination modified Fe 3 O 4 Nanoparticles. 1mL of aminated Fe was taken 3 O 4 Adding 10mL of Au nanoparticle solution into the solution, placing the solution on a horizontal shaker at room temperature for reaction overnight, and washing the solid product with ethanol and water for three times respectively to obtain Au @ Fe 3 O 4 Nanoparticles.
(4)Au@Fe 3 O 4 Preparation of-substrate-aptamer Probe
1mL 1mg/mL Au @ Fe 3 O 4 Mixing the nanoparticle water solution with 400uL 1 μ M DNA substrate solution, reacting at 37 deg.C for 24 hr, and magnetically treatingAfter sexual isolation, the solid complex was washed three times with water and the product was resuspended in 1mL of PBS buffer (Ph7.4) to give Au @ Fe 3 O 4 -a substrate solution. Then mixing the above solution with 400uL 1 μ M aptazyme, reacting at 37 deg.C overnight, magnetically separating the solid product, washing with water three times to obtain Au @ Fe 3 O 4 -substrate-aptazyme probe.
Example 2
RuSiO 2 The CS solution was prepared as follows:
2mL of Triton X-100,8mL of cyclohexane and 2mL of n-hexanol were weighed and added to a reaction vessel, and after thorough mixing, 350. Mu.L of 40mM Ru (bpy) was added to the mixture 3 Cl 2 Uniformly mixing the aqueous solution, adding 150 mu L TEOS and 100 mu L ammonia water, and stirring to react for 24h; adding 5mL of acetone, centrifuging, washing the precipitate with ethanol and water in sequence, and resuspending the product with ethanol to obtain 4mg/mL RuSiO 2 And (3) solution.
Adding 1mg of CS into 1mL of acetic acid aqueous solution with the volume fraction of 2%, and performing ultrasonic dispersion for 20min to obtain a CS solution; take 1mL 4mg/mL RuSiO 2 Adding the aqueous solution into 1mL of CS solution, and carrying out ultrasonic treatment for 30min to obtain RuSiO with uniform dispersion 2 -a CS solution.
Example 3
Preparation method of electrochemiluminescence sensor for measuring Trigger DNA
The preparation method of the electrochemical luminescence sensor is shown in figure 1 and comprises the following steps:
(1) Electrode pretreatment: the working electrode was successively treated with 0.3 μm and 0.05 μm Al 2 O 3 Polishing the powder, and then ultrasonically cleaning for 4min by using water, ethanol and water in sequence;
(2) Modified RuSiO 2 : taking 10 mu L of 2mg/mL RuSiO 2 -CS solution (example 2) was added dropwise to the surface of the working electrode, left to stand at room temperature for drying, washed with PBS solution, and dried;
(3) Covalent attachment of Capture DNA: dripping 2.5% glutaraldehyde aqueous solution on the surface of the electrode, reacting for 2h at room temperature, cleaning with PBS solution, and air drying; dripping 10 mu L of 4 mu M Capture DNA on the surface of an electrode, reacting for 2h at 37 ℃, washing with a PBS solution, and drying in the air;
(4) And (3) sealing: 10 mu L of 4 mu M Blocker is dripped on the surface of the electrode, and the reaction is carried out for 2h at room temperature, washed by PBS solution and dried.
Example 4
Cyclic voltammetry monitoring of electrochemical sensor assembly process
To investigate the successful preparation of the electrochemiluminescence sensor by performing cyclic voltammetric scanning to record the signal response analysis of the sensor at each modification stage, the glassy carbon electrode (working electrode) obtained from each step in example 3 was placed in a solution containing 2mM K 3 [Fe(CN) 6 ]In 0.01M PBS at a rate of 0.1V/s, and the results are shown in FIG. 2. When RuSiO 2 After CS is modified on the surface of the electrode, the peak current value of the CV curve is reduced compared with that of a bare electrode due to the increase of the resistance. After the step of the Capture DNA modification, the current value is obviously reduced, which indicates that a large amount of Capture DNA is modified on the surface of the electrode, and is beneficial to the smooth operation of the subsequent antigen detection step. Then, the working electrode undergoes small-amplitude reduction of current every time the working electrode undergoes modification, which is caused by steric hindrance and electronegativity generated after DNA is combined on the surface of the electrode. This example illustrates the use of electrochemical methods to monitor the sensor assembly process, which illustrates the successful modification of the corresponding materials and DNA to the electrode surface during the sensor fabrication process.
Example 5
Electrochemiluminescence sensor system detection process for detecting adenosine triphosphate
(1) Taking 100 μ L ATP solution to be detected, and mixing with 100 μ L Au @ Fe 3 O 4 Mixing a substrate-aptamer probe, reacting at 37 ℃ for 2h, and performing magnetic separation;
(2) mu.L of digestion buffer (containing 100mM NaCl and 20mM MgCl) 2 20mM HEPES buffer solution) resuspending the adenosine triphosphate-adsorbing magnetic probe in the step (1), reacting for 2h at 37 ℃, and magnetically separating;
(3) Mixing the supernatant containing Trigger DNA with 2 mu M hairpin-Fc solution in equal volume, dripping 10 mu L of the mixture on the surface of an electrode, reacting for 2h at 37 ℃, washing with PBS solution, and drying in the air;
(4) ECL luminescence signal of the test sensor: the sensor is placed in 0.01M PBS (pH7.4) solution containing 25mM triethylamine for CV test, the potential range is 0-1.3V, the speed is 0.1V/s, ECL luminescence signals are synchronously recorded, the photomultiplier high Pressure (PMT) is 800V, the attenuation value of the ECL signals is calculated, and the corresponding concentration of the adenosine triphosphate is calculated according to a standard curve.
Example 6
Optimization of incubation time of electrochemiluminescence sensor system for measuring adenosine triphosphate
The effect of incubation time of Trigger and Hairpi-Fc on ECL signal intensity on the electrode surface was explored. The detection process of the electrochemiluminescence sensor system is the same as that of the electrochemiluminescence sensor system in example 5, the ATP concentration is 100pM, and 5 different times of incubation of the mixed solution containing the supernatant of Trigger DNA and the Hairpin-Fc on the surface of the electrode in the step (3) are selected from 30min, 60min, 90min, 120min, 150min and the like. As shown in FIG. 3, when the reaction time is less than or equal to 90min, the Δ ECL signal intensity increases rapidly with the time, and reaches the maximum value at 90min, so that the electrochemiluminescence sensor system of the present invention selects 90min as the incubation time.
Example 7
Linear relationship between delta ECL intensity and antigen concentration of electrochemical luminescence sensor system for measuring adenosine triphosphate
The procedure of the electrochemiluminescence sensor system was the same as in example 5. Various concentrations of adenosine triphosphate standard solutions were prepared, 0.1pM,1pM,10pM,100pM,1000pM, and 3 parallel experimental groups were set at each concentration, and the detection method of example 5 was used.
The linear relationship between the Δ ECL signal intensity and the antigen concentration was analyzed and the results are shown in fig. 4. The Δ ECL signal intensity gradually increases with increasing ATP concentration and is linear. The lowest detection limit of the sensor is 0.054pM, and compared with the existing commercial ATP detection kit (chemiluminescence method, the lowest detection limit is 0.1 nM), the invention has higher sensitivity.
Example 8
Specific analysis of target detection by electrochemiluminescence sensor system for measuring adenosine triphosphate
Examining whether the electrochemiluminescence sensor system has nonspecific response to the structural analog of the target analyte, the detection process of the electrochemiluminescence sensor system is the same as that in example 5, except that different antigens are selected in the step (1): UTP, GTP, CTP instead of ATP, the results are shown in FIG. 5. Under the same concentration, namely 1000pM, the sensor can effectively distinguish ATP, and when the structural analogue exists, the response signal of the sensor has no obvious change compared with a control group, which shows that the sensor designed by the invention has good specificity.
Example 9
The electrochemical luminescence sensor system of the invention applies the quantitative detection of ATP in human serum
In order to examine the feasibility of the electrochemiluminescence sensor system for quantitatively detecting ATP in human serum, the detection process of the electrochemiluminescence sensor system is the same as that of example 5, and the preparation method of the ATP solution to be detected used in the step (1) comprises the following steps: different amounts of ATP were weighed and dissolved in 10% volume fraction human serum solution (solvent 0.01M PBS, pH 7.4) to final concentrations of 0.1pM,1pM,10pM,100pM,1000pM, respectively. The result is shown in fig. 6, and the detection result shows that the recovery rate of ATP in human serum detected by the electrochemiluminescence sensor system disclosed by the invention is distributed in 95.68% -103.4%, and the relative standard deviation between different control groups is distributed in 1.881% -5.689%, which indicates that the sensor system has stable test data and good reliability, and can be used for quantitative detection of ATP in human serum.
Example 10
The preparation of the Au @ Fe3O4-substrate-aptamer probe in the embodiment is the same as the preparation method of the embodiment 1, except that: in the step (3), the addition amount of the APTES is 10 percent of the volume of the reaction solution; fe 3 O 4 The concentration of the nano particles is 5mg/mL; the concentration of the Au @ Fe3O4 nano particles in the step (4) is 5mg/mL, the concentration of DNA superstrate is 10 muM, and the concentration of aptazyme is 10 muM.
RuSiO 2 The CS solution was prepared as in example 2, except that: measuring 1mL of Triton X-100,5mL of cyclohexane, adding 1mL of hexanol into the mixture for reactionAfter thoroughly mixing in a container, 250. Mu.L of 5mM Ru (bpy) was added to the mixture 3 Cl 2 Mixing the aqueous solution evenly, adding 50 mu L TEOS and 30 mu L ammonia water, and stirring for reaction for 12 hours; adding 1mL of acetone, centrifuging, sequentially cleaning precipitates with ethanol and water, and resuspending the product with ethanol to obtain 1mg/mL RuSiO 2 And (3) solution.
Adding 0.1mg of CS into 0.1mL of acetic acid aqueous solution with volume fraction of 0.5%, and performing ultrasonic dispersion for 5min to obtain a CS solution; take 0.1mL 1mg/mL RuSiO 2 Adding the aqueous solution into 0.1mL of CS solution, and carrying out ultrasonic treatment for 10min to obtain RuSiO with uniform dispersion 2 -a CS solution.
An electrochemiluminescence sensor was prepared as in example 2, except that: (1) electrode pretreatment: the working electrode was successively treated with 0.3 μm and 0.05 μm Al 2 O 3 Polishing the powder, and then carrying out ultrasonic cleaning for 2min by using water, ethanol and water in sequence;
(2) Modified RuSiO 2 : 2 μ L of 1mg/mL RuSiO 2 Dripping a CS solution on the surface of the working electrode, standing and drying at room temperature, washing with a PBS solution, and drying in the air;
(3) Covalent attachment of Capture DNA: dripping glutaraldehyde aqueous solution with the mass fraction of 0.5% on the surface of the electrode, reacting for 1 hour at room temperature, cleaning with PBS solution, and drying; dripping 2 mu L of 1 mu M Capture DNA on the surface of an electrode, reacting for 1h at 37 ℃, washing with a PBS solution, and drying in the air;
(4) And (3) sealing: and (3) dropwise adding 2 mu L of 1 mu M Blocker on the surface of the electrode, reacting at room temperature for 1h, washing with a PBS solution, and drying.
Example 11
Example 10 was prepared in the same manner as example 1, except that: in the step (3), the addition amount of the APTES is 5 percent of the volume of the reaction solution; fe 3 O 4 The concentration of the nano particles is 0.5mg/mL; the concentration of the Au @ Fe3O4 nano particles in the step (4) is 0.5mg/mL, the concentration of DNA superstrate is 5 mu M, and the concentration of aptazyme is 5 mu M.
RuSiO 2 The CS solution was prepared as in example 2, except that: measuring 3mL of Triton X-100, 10mL of cyclohexane and 3mL of hexanol, adding the mixture into a reaction vessel, fully mixing,to the mixture was added 500. Mu.L of 100mM Ru (bpy) 3 Cl 2 Mixing the aqueous solution evenly, adding 200 mu L TEOS and 200 mu L ammonia water, and stirring for reacting for 36h; adding 10mL of acetone, centrifuging, sequentially cleaning precipitates with ethanol and water, and resuspending the product with ethanol to obtain 8mg/mL RuSiO 2 And (3) solution.
Adding 2mg of CS into 2mL of acetic acid aqueous solution with volume fraction of 3%, and performing ultrasonic dispersion for 5min to obtain a CS solution; take 2mL 8mg/mL RuSiO 2 Adding the aqueous solution into 2mL of CS solution, and carrying out ultrasonic treatment for 60min to obtain RuSiO with uniform dispersion 2 -a CS solution.
An electrochemiluminescence sensor was prepared as in example 2, except that: (1) electrode pretreatment: the working electrode was successively treated with 0.3 μm and 0.05 μm Al 2 O 3 Polishing the powder, and then carrying out ultrasonic cleaning for 10min by using water, ethanol and water in sequence;
(2) Modified RuSiO 2 : 20 mu L of RuSiO 8mg/mL is taken 2 Dripping a CS solution on the surface of the working electrode, standing and drying at room temperature, washing with a PBS solution, and drying in the air;
(3) Covalent attachment of Capture DNA: dropwise adding a glutaraldehyde aqueous solution with the mass fraction of 5% to the surface of the electrode, reacting for 3 hours at room temperature, cleaning with a PBS solution, and drying in the air; dripping 20 mu L of 10 mu M Capture DNA on the surface of an electrode, reacting for 3h at 37 ℃, washing with a PBS solution, and drying in the air;
(4) And (3) sealing: 20 μ L of 10 μ M Blocker was dropped on the electrode surface, and the reaction was carried out at room temperature for 3 hours, washed with PBS solution, and dried.
Sequence listing
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Claims (6)

1. An electrochemical luminescence sensor system for measuring adenosine triphosphate is characterized in that the system is composed of a magnetic probe Au @ Fe for amplifying an adenosine triphosphate concentration signal 3 O 4 -substrate-aptazyme, electrochemiluminescence sensor for measuring Trigger DNA and hairpin-Fc; the magnetic probe Au @ Fe 3 O 4 -substrate-aptazyme is derived from Fe 3 O 4 NanoparticlesGold nanoparticles, DNA substrate and aptazyme are compounded in sequence; the electrochemiluminescence sensor for measuring Trigger DNA connects amino modified Capture DNA to modified RuSiO through glutaraldehyde crosslinking 2 -the working electrode surface of CS is obtained;
the hairpin-Fc sequence is TCGTAGCAATACCAGGAGGCTACGATGACTCACTCCTGGTAT-Fc;
the DNA substrate sequence is SH-AAAAAAAATTCACCAACTAATrAGCT ACGATGACTCACCTAGGAG; the aptazyme sequence is 'TCATCGTAGAGCGAT CTAGGGGGAGTATTGCGGAGGATATCACCCATGTTAGTTGGTAA'; the amino modified Capture DNA sequence is NH 2 -CTCCTAGGTGAGTCATCGTAGCCTCCTGGTATTG CTACGATGACTCA；
The magnetic probe Au @ Fe 3 O 4 The substrate-aptamer preparation procedure is as follows:
(1) Weighing FeCl 3 And dissolving trisodium citrate in ethylene glycol, adding sodium acetate, adding the stirred mixed solution into a reaction kettle, and cleaning the precipitate after reaction to obtain Fe 3 O 4 Nanoparticles;
(2) To HAuCl 4 Adding trisodium citrate aqueous solution into the aqueous solution, and continuously boiling to obtain Au nanoparticles;
(3) Using APTES on Fe 3 O 4 Performing amination modification on the nanoparticles, mixing the product with Au nanoparticles, performing magnetic separation, and cleaning to obtain Au @ Fe 3 O 4 Nanoparticles;
（4）Au@Fe 3 O 4 mixing the nano particles and DNA substrate, magnetically separating after reaction, and washing with water to obtain Au @ Fe 3 O 4 -a substrate solution; then mixed with aptazyme for overnight reaction to obtain Au @ Fe 3 O 4 -a substrate-aptazyme probe;
the preparation process of the electrochemiluminescence sensor for determining Trigger DNA is as follows:
(1) Polishing the working electrode, and then ultrasonically cleaning;
(2) Taking RuSiO 2 Dripping a CS solution on the surface of the working electrode, standing and drying at room temperature, and cleaning and airing;
(3) Dripping glutaraldehyde aqueous solution on the surface of the electrode, reacting at room temperature, and then cleaning and airing; dropwise adding the Capture DNA to the surface of an electrode, and cleaning and airing after reaction;
(4) And dropwise adding Blocker on the surface of the electrode, reacting at room temperature, cleaning and airing to obtain the electrochemiluminescence sensor for measuring Trigger DNA.
2. The electrochemiluminescence sensor system for detecting adenosine triphosphate according to claim 1, wherein the Trigger DNA sequence is GCTACGATGACTCACCTAGGAG.
3. The electrochemiluminescence sensor system for detecting adenosine triphosphate according to claim 1, wherein the RuSiO is 2 -CS is from RuSiO 2 Mixing the solution with a solution containing CS; the RuSiO 2 Adding Ru (bpy) into a mixture of Triton X-100, cyclohexane and n-hexanol 3 Cl 2 Mixing the aqueous solution, adding TEOS and ammonia water, and stirring for reaction; adding acetone, centrifuging, cleaning and precipitating to obtain RuSiO 2 。
4. The electrochemiluminescence sensor system for detecting adenosine triphosphate according to claim 1, wherein the magnetic probe au @ fe 3 O 4 In the step (3) of preparing substrate-aptazyme, the amount of APTES added is 1 to 10% of the reaction solution, and Fe 3 O 4 The concentration of the nano particles is 0.5 to 5mg/mL; step (4) the Au @ Fe 3 O 4 The concentration of the nano particles is 0.5 to 5mg/mL, the concentration of DNA substrate is 1 to 10 mu M, and the concentration of aptazyme is 1 to 10 mu M.
5. Use of an electrochemiluminescence sensor system for the determination of adenosine triphosphate according to claim 1 for the quantitative detection of adenosine triphosphate, said use comprising the steps of detecting adenosine triphosphate as follows:
(1) Adenosine triphosphate solution and magnetic probe Au @ Fe 3 O 4 -substrate-aptaMixing and continuously reacting zyme, and separating a binding compound of the adenosine triphosphate and the magnetic probe by using magnetic adsorption;
(2) Resuspending the compound by using enzyme digestion buffer solution, activating enzyme digestion reaction to generate a large amount of Trigger DNA, and removing the magnetic probe by magnetic adsorption to obtain supernatant solution containing the Trigger DNA;
(3) And (3) mixing the solution with the hairpin-Fc in the same volume, dripping the mixture on the surface of the electrochemical luminescence sensor, reacting for a period of time, testing an electrochemical luminescence signal of the sensor, and calculating the corresponding ATP concentration according to the attenuation value of the electrochemical luminescence signal.
6. Use according to claim 5, characterized in that it is a magnetic probe Au @ Fe 3 O 4 -substrate-aptazyme is used for specific capture of adenosine triphosphate molecules, and simultaneously starting isothermal enzyme amplification reaction of aptazyme to generate a large amount of Trigger DNA; under the catalytic action of Trigger DNA, hairpin-Fc and RuSiO on the surface of the sensor 2 -complementary binding of the modified Capture DNA on CS, thereby immobilizing a plurality of hairpin-Fc on the electrode surface; dose-dependent inhibition of RuSiO due to Fc 2 The electrochemical luminescence signal of CS, so that the concentration of the adenosine triphosphate and the attenuation value of the electrochemical luminescence signal are in a linear relation to realize the quantitative detection of the adenosine triphosphate.
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