CN109738495B - Tri-metal signal amplification aptamer sensor based on cerium metal organic framework @ gold nanocomposite and gold platinum ruthenium nanocomposite for detecting thrombin-sensitive protein-1 - Google Patents
Tri-metal signal amplification aptamer sensor based on cerium metal organic framework @ gold nanocomposite and gold platinum ruthenium nanocomposite for detecting thrombin-sensitive protein-1 Download PDFInfo
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Abstract
The invention uses an aptamer as a target capture agent, and successfully develops a sensor for sensitive detection of thrombin sensitive protein-1 (TSP-1) based on a signal amplification strategy of a cerium metal organic framework @ gold nanocomposite (Ce-MOF @ Au) and a gold platinum ruthenium nanocomposite (AuPtRu NP). The synthesized AuPtRu NP can be used as a catalyst for catalyzing hydrogen peroxide, and can also be used as a nano carrier for capturing amino (-NH2) to terminate single-stranded DNA (S1) so as to obtain a signal probe SP (AuPtRu NP/S1). The Ce-MOF @ Au is obtained by in-situ reduction and is used as an electrode modification material of a glassy carbon electrode. When the detection solution contains H2O2The AuPtRu NP can oxidize H2O2To obtain an enhanced signal. The present invention shows from 1fg mL‑1To 10ng mL‑1Has a linear range of 0.13fg mL‑1Very low detection limit. In addition, the aptamer sensor can be directly used for detecting clinical practical samples.
Description
The technical field is as follows:
the invention relates to a preparation method and application of an electrochemical aptamer sensor for clinically and quantitatively detecting thrombin-sensitive protein-1, in particular to a biosensor prepared by taking a cerium metal organic framework @ gold nanocomposite (Ce-MOF @ Au) and a gold platinum ruthenium nanocomposite (AuPtRu NP) as a signal probe, which is used for detecting thrombin-sensitive protein-1 and belongs to the field of electrochemical detection.
Background art:
cardiovascular disease (CVD) is a disease of the heart and blood vessels and is a leading cause of death worldwide. Individuals at risk for CVD may exhibit an increase in the level of biomarker molecules in the blood. Identifying the highest CVD risk biomarker for aiding diagnosis may prevent premature death. Thrombospondin-1 (TSP-1) is a promising candidate biomarker for CVD, and is highly expressed in large atherosclerotic lesions and myocardial infarction. Therefore, a series of methods, such as tetrazolium salt colorimetry and enzyme-linked immunosorbent assay (ELISA), have been developed for detecting TSP-1. However, all of these methods require complicated processing of the target sample. Thus, sensitive and selective detection of TSP-1 in clinical samples remains challenging. Due to its extremely low detection limit and potential for in situ analysis, electrochemical sensors enable TSP-1 detection for use in CVD diagnostics.
In recent years, researchers have attracted attention to Metal Organic Frameworks (MOFs), which are novel microporous materials having metal ions as nodes and organic ligands as linkers. MOFs have adjustable size and morphology, have high porosity, satisfactory electrochemical stability, and are easily modified. Inspired by the characteristics of MOFs, cerium (Ce) -based MOFs (Ce-MOFs) were synthesized in this work, which not only inherit the advantages of traditional MOFs, but also show strong loading capacity and good biocompatibility. The advantages of Ce-MOF indicate that Ce-MOF is very suitable for use as an electrode modifying material. We found that the synthesis of Ce-MOF using trimesic acid as organic framework has the advantages of high efficiency and simple steps, which undoubtedly will increase the feasibility of sensor construction. In order to increase the sensitivity of the sensor and immobilize more capture probe dna (CP), gold nanoparticles (AuNPs) were modified onto the surface of the prepared Ce-MOF to act as nanocarriers, and amino-modified CP was immobilized by gold-ammonia bonds. In our previous work, we found that gold tends to aggregate when reduced in situ to the support surface using a reducing agent, which makes the synthesis process extremely limited. It is reported that PVP molecules can adsorb on the growing noble metal core as a typical surfactant while preventing aggregation of the noble metal nanoparticles. To solve this problem, polyvinylpyrrolidone (PVP) was used as a protectant in this work, successfully avoiding the aggregation of AuNPs. To our knowledge, this was the first development of a one-step process to reduce Au NPs to the surface of Ce-MOFs in situ to form stable nanocomposites (Ce-MOF @ Au) and to be used as electrode modification materials.
The use of conventional biological recognition molecules (e.g., enzymes, antibodies) in biosensors is limited due to their instability. Aptamers, while having high affinity and specificity for a variety of target substances from small molecules to large proteins, have been increasingly used in a variety of sensing platforms. Aptamers are short synthetic nucleotide sequences obtained by in vitro methods from cell SELEX. In addition to their affinity for targets, aptamers also have advantages such as ease of production, low cost synthesis, high thermal stability, and ease of labeling and modification. They can also be easily immobilized on different surfaces with various chemistries. Therefore, aptamers are applied as specific recognition substances in various sensors as substitutes for antibodies. Our goal was to design an aptamer sensor that combines the specificity of the aptamer with a signal amplification mechanism for TSP-1 detection.
To achieve sensitive detection of trace species, secondary signal species are commonly used to amplify the electrochemical response in electrochemical sensor analysis techniques. Platinum (Pt) nanoparticles have long been known to have good catalytic properties, and reasonable composition and structure are critical to the design of high activity platinum catalysts. Currently, more and more platinum trimetallic materials, such as FePtCu, AuPdPt and PtPdTe, have been synthesized, and the catalytic capability of the materials is superior to that of single platinum and platinum bimetallic materials. Furthermore, it is known that ruthenium (Ru) metal is very stable and can be used as an excellent catalyst. Further, ruthenium is an effective hardener for platinum, and can be used to improve the stability and catalytic performance of platinum. The Au NPs have good chemical stability and biocompatibility, and are widely applied to electrochemical signal materials. Therefore, the research combines the advantages of Au NPs, Pt NPs and Ru NPs, and the AuPtRu trimetal nano compound (AuPtRu NPs) is designed and synthesized for the first time. The AuPtRu NP has good stability, catalytic performance and capability of fixing biological molecules. Briefly, H is coupled by AuPtRu NPs2O2The electrochemical signal is obviously amplified by the concerted catalysis of the (C).
In order to construct a multi-element, synergistic nano-sensing interface, a modified amino (-NH) group at the 5' end is designed2) The single-stranded nucleic acid 1(S1) is reacted with an aptamer to form a double strand in a PCR machine. Then, S1 was performed by Au-NH2Bond and Pt-NH2The bond was attached to the signaling material AuPtRu NPs, forming a Signaling Probe (SP). TSP-1 binds to aptamers and induces SP from M1(SP + aptamers) in the presence of TSP-1Body) is released freely. At the same time, reacting-NH2A modified Capture Probe (CP) was immobilized on the electrode modified with Ce-MOF @ Au. AuPtRu catalyzes H when CP recognizes released SPs and captures them2O2A large electrical signal is generated. The Ce-MOF @ Au has excellent conductivity and the excellent catalytic capability of AuPtRu NPs, so that the novel technical platform realizes the sensitive detection of TSP-1 in serum. We prove that the proposed aptamer sensor has good application prospects in clinical research.
The project establishes a simple and rapid detection method to realize the specific and ultrasensitive detection of the TSP-1. Provides basis for early detection and risk prediction of patients with cardiovascular diseases.
The invention content is as follows:
1. the invention aims to provide a preparation method and application of an electrochemical aptamer sensor for detecting thrombospondin-1, which provides a basis for early detection and risk prediction of patients with cardiovascular diseases clinically, and is characterized by comprising the following steps of:
(1) preparing a cerium metal organic framework @ gold nanocomposite (Ce-MOF @ Au) and a gold platinum ruthenium nanocomposite (AuPtRu NPs) signal probe;
(2) and establishing an electrochemical aptamer sensor, detecting the thrombin sensitive protein-1, and drawing a standard curve.
2. The preparation process of the cerium metal organic framework @ gold nanocomposite and the gold platinum ruthenium nanocomposite signal probe specifically comprises the following steps:
(1) preparation of the Ce-MOF @ Au composite material:
first, 4.34g of Ce (NO) was added3)3·6H2O was added to 45mL of ultrapure water and dissolved to form solution A. Then 10mL of water-ethanol (1: 1) was used as solvent, and 2.10g of H was added3BTC, forming solution B. The solution A was gradually added dropwise to the solution B under vigorous stirring (800rpm) at 60 ℃ and reacted sufficiently for one hour. Centrifuging (8000rpm), washing with ethanol and ultrapure water for several times to obtain Ce-MOF, and drying for use. Then preparing the nano composite material Ce-MOF @ Au, firstly weighing 1mg of Ce-MOF to be dispersed in 2mLUltrapure Water, then 2mL of 2% HAuCl was added4After mixing well, 2mL of PVP solution (2mg mL) was added-1) And ultrasonically mixing for 15 min. Stirring at 800rpm, slowly adding 2mL NaBH dropwise4(7.5mg mL-1) And continuously stirring for 30min at room temperature, centrifuging, washing with ultrapure water for three times to obtain the Ce-MOF @ Au nano composite, and dispersing in Milli-Q water for further use.
(2) Preparation of gold platinum ruthenium trimetal nanoparticles (AuPtRu NPs):
first, 0.9mL of 20mM RuCl3、53.2μL 5％HAuCl4、88.4uL 5％H2PtCl6And 0.01g of Pluronic F127 were placed in a beaker and mixed well. Then 0.3mL of 0.4M AA was added dropwise with stirring (800rpm) and stirring was continued at room temperature for 3 hours. Centrifuged, washed three times with ultrapure water, and dispersed in 500uL of ultrapure water.
(3) Preparation of Signaling Probe (SP):
first, a 4. mu.M signal strand (S1) was mixed with a 4. mu.M aptamer strand in equal volumes, heated at 90 ℃ for 10 minutes, and then cooled to room temperature for 1 hour to give a partially hybridized double strand. mu.L of the double strand formed by hybridization was added to 1mL of the AuPtRu trimetal mixed solution, shaken at 4 ℃ for 12 hours, the mixture was centrifuged, washed twice with water (8000rpm), dissolved in 1mL of the hybridization solution, and then 0.25% BSA was added thereto to block the nonspecific binding site, shaken at 4 ℃ for 2 hours, centrifuged again (5000rpm), washed once with water, and dispersed in 1mL of the hybridization solution. Finally, 200. mu.L of thrombin-sensitive protein-1 was added to the mixture obtained above at various concentrations to dissociate the signal chain in the double strand, thereby obtaining an electrochemically active Signal Probe (SP).
3. The method for establishing an electrochemical aptamer sensor according to claim 1, detecting thrombospondin-1, and drawing a standard curve, which is characterized by comprising the following steps:
(1) with 0.3 and 0.05 μm Al, respectively2O3Polishing the electrode into a mirror surface by using powder, then respectively carrying out ultrasonic treatment on the electrode for 5min according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;
(2) and dripping 8 mu L of electrode modification material cerium metal organic framework @ gold (Ce-MOF @ Au) nano composite on the surface of the electrode, and drying at room temperature.
(3) 10uL of 4. mu.M Capture Probe (CP) solution was bound to the dried electrode surface (37 ℃, 2.5 h).
(4) After the incubated electrode was washed clean with ultrapure water, 6. mu.L of 0.5% BSA solution was added dropwise and incubated at room temperature for 30 min.
(5) After the electrode was rinsed with ultrapure water, 10. mu.L of the prepared Signal Probe (SP) was dropped on the electrode and incubated at 37 ℃ for 2 h.
(6) And washing the incubated electrode with ultrapure water, and drying at room temperature.
(7) The electrode was placed in 5mL, 0.1M PBS (0.1M Na)2HPO4，0.1M KH2PO40.1M KCI) with 20. mu.L of 2.2mM H added every 50s2O2And measuring the current value of the timing current change.
(8) And drawing a working curve according to the linear relation between the obtained current change value and the concentration of the thrombin sensitive protein-1.
Compared with the prior art, the preparation method and the application of the electrochemical aptamer sensor for quantitatively detecting the TSP-1 have the outstanding characteristics that:
(1) the cerium metal organic framework @ gold nanocomposite (Ce-MOF @ Au) is used as an electrode modification material, and the gold platinum ruthenium nanocomposite is used as a signal probe, so that the catalytic performance of the material is effectively improved, the solid loading capacity of biomolecules is improved, and the sensitivity and the detection range of the electrochemical aptamer sensor are further improved;
(2) the electrochemical ultra-sensitive detection method can be used for detecting TSP-1 in real clinical samples.
(3) The electrochemical aptamer sensor prepared by the method can provide basis for clinical early diagnosis of cardiovascular disease patients, and can also be used for predicting the risk of occurrence of cardiovascular events. In addition, the method is simple, convenient and quick, and is convenient for realizing commercialization, thereby promoting the development of precise medicine.
(4) By using the completely same nano material and modification method, different aptamers and corresponding complementary chains can be used, so that simultaneous detection of various biomolecules is realized, and a more comprehensive basis is provided for diagnosis of diseases.
Description of the drawings:
FIG. 1 is a schematic diagram of the construction of an electrochemical aptamer sensor according to the invention.
FIG. 2 is a graph showing a comparative electron microscope, DPV, XPS and UV absorption of the cerium metal organic framework and the cerium metal organic framework @ gold nanocomposite (Ce-MOF @ Au) according to the present invention.
FIG. 3 is the electron microscope image, zeta potential image and timing current image of the Au/Pt/Ru nanocomposite material of the present invention.
FIG. 4 is an atomic force microscope and electrochemical characterization of the electrode construction process of the present invention.
Fig. 5 is a linear relationship between the chronoamperometric change current and the concentration of the electrochemical aptamer sensor of the invention when detecting TSP-1, and the reproducibility and specificity of the sensor.
The specific implementation mode is as follows:
the invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
Example 1
and 3, dripping 8 mu L of electrode modification material cerium metal organic framework @ gold nano composite (Ce-MOF @ Au) on the surface of the electrode, and drying at room temperature.
Step 4. bind 10uL of 4. mu.M Capture Probe (CP) solution to the dried electrode surface (37 ℃, 2.5 h).
And 5, washing the incubated electrode with ultrapure water, and then dropwise adding 6 mu L of 0.5% BSA solution for incubation at room temperature for 30 min.
And 6, washing the electrode with ultrapure water, dropwise adding 10 mu L of the prepared Signal Probe (SP) on the electrode, and incubating for 2h at 37 ℃.
And 7, washing the incubated electrode with ultrapure water, and drying at room temperature.
Step 8. Place the electrode in 5mL, 0.1M PBS (0.1M Na)2HPO4，0.1M KH2PO40.1M KCI) with 20. mu.L of 2.2mM H added every 50s2O2Measuring the timing current change current value;
step 9, drawing a working curve according to the linear relation between the obtained current change value and the concentration of the thrombin sensitive protein-1 (TSP-1); the measurement result shows that the concentration of the TSP-1 is 1fg mL-1-10ng mL-1Linear relation is formed in the range, the linear correlation coefficient is 0.9975, and the detection limit is 0.13fg mL-1。
Step 11, storing the sensor at 4 ℃, intermittently detecting the current response of the sensor, and after storing for 30 days, the current response is still 91.60 percent of the initial current, which indicates that the sensor has excellent stability;
And step 13, detecting the TSP-1 by using the sensor in the presence of other biomolecules in blood, wherein the change of the TSP-1 current is not influenced by the presence of other biomolecules, which shows that the specificity of the sensor is good, and target detection objects can be well distinguished.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (2)
1. A preparation method of an electrochemical aptamer sensor constructed on the basis of a cerium metal organic framework @ gold nano composite and a gold platinum ruthenium trimetal signal probe is characterized by comprising the following steps:
(1) preparing a cerium metal organic framework @ gold nano composite:
first, 4.34g of Ce (NO) was added3)3·6H2Adding O into 45mL of ultrapure water, and dissolving to form a solution A; then, mixing 10mL of the mixture in a volume ratio of 1: 1 water-ethanol as solvent, and 2.10g of H3BTC, forming solution B; gradually dropwise adding the solution A into the solution B under the conditions of 60 ℃ and intensive stirring at 800rpm, and fully reacting for one hour; then, centrifugal separation is carried out at 8000rpm, and the mixture is washed for a plurality of times by ethanol and ultrapure water to obtain Ce-MOF, and the Ce-MOF is dried for standby; then preparing a cerium metal organic framework @ gold nano composite by firstly weighing 1mg of Ce-MOF and dispersing in 2mL of ultrapure water, and then adding 2mL of 2% HAuCl4After fully mixing, 2mL and 2mg mL of the mixture was added-1Carrying out ultrasonic mixing on the PVP solution for 15 min; stirring at 800rpm, 2mL of 7.5mg mL was slowly added dropwise-1NaBH of4Continuously stirring the solution at room temperature for 30min, centrifuging, washing with ultrapure water for three times to obtain a cerium metal organic framework @ gold nano composite, and dispersing in Milli-Q water for further use;
(2) preparing a gold-platinum-ruthenium trimetal signal probe;
first, 0.9mL of 20mM RuCl3、53.2μL 5％HAuCl4、88.4uL 5％H2PtCl6And 0.01g of Pluronic F127 are put into a beaker and mixed evenly; then gradually dropwise adding 0.3mL of 0.4M ascorbic acid under stirring at 800rpm, and continuously stirring for 3 hours at room temperature; centrifuging, washing with ultrapure water for three times, and dispersing in 500uL of ultrapure water for later use; then mixing the 4 mu M signal chain and the 4 mu M aptamer chain in equal volume, heating the mixture at 90 ℃ for 10 minutes, and then cooling the mixture to room temperature for 1 hour to obtain a partially hybridized double chain; adding 200 mu L of double chains formed by hybridization into 1mL of gold platinum ruthenium trimetal mixed solution, oscillating for 12 hours at 4 ℃, centrifuging the mixture at 8000rpm, washing twice, dissolving in 1mL of hybridization solution, then adding 0.25% BSA (bovine serum albumin) to block non-specific binding sites, oscillating for 2 hours at 4 ℃, centrifuging at 5000rpm again, washing once, and dispersing in 1mL of hybridization solution; finally, 200 mu L of thrombin sensitive protein-1 with different concentrations is added into the obtained mixed liquid, so that a signal chain in a double chain is dissociated, and the gold platinum ruthenium trimetal signal probe with electrochemical activity is obtained;
(3) the electrochemical aptamer sensor is constructed by adopting the composite material and the signal probe.
2. The electrochemical aptamer sensor obtained by the preparation method according to claim 1, which is used for detecting thrombospondin-1, and is characterized in that the detection steps are as follows:
(1) with 0.3 and 0.05 μm Al, respectively2O3Polishing the electrode into a mirror surface by using powder, then respectively carrying out ultrasonic treatment on the electrode for 5min according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;
(2) dripping 8 mu L of electrode modification material cerium metal organic framework @ gold nano composite on the surface of an electrode, and drying at room temperature;
(3) binding 10uL of 4. mu.M capture probe solution to the dried electrode surface at 37 ℃ for 2.5 h;
(4) rinsing the incubated electrode with ultrapure water, and then dropwise adding 6 mu L of 0.5% BSA solution for incubation at room temperature for 30 min;
(5) washing the electrode with ultrapure water, dripping 10 mu L of the prepared signal probe on the electrode, and incubating for 2h at 37 ℃;
(6) rinsing the incubated electrode with ultrapure water, and drying at room temperature;
(7) the electrode was placed in 5mL of 0.1M Na2HPO4,0.1M KH2PO4Characterization was performed in 0.1M PBS prepared with 0.1M KCl, and 20. mu.L of 2.2mM H was added every 50s2O2Measuring the timing current change current value;
(8) and drawing a working curve according to the linear relation between the obtained current change value and the concentration of the thrombin sensitive protein-1.
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