Thrombin aptamer sensor based on Ti3C2 and preparation method of thrombin aptamer sensor

An aptamer sensor and thrombin technology, applied in instruments, scientific instruments, fluorescence/phosphorescence, etc., can solve the problems of complex and time-consuming reactions, difficult preparation and storage of antibodies, etc., and achieve the effect of fast reaction, easy storage, and simple synthesis

Active Publication Date: 2020-03-24
JILIN UNIV
9 Cites 0 Cited by

AI-Extracted Technical Summary

Problems solved by technology

However, the disadvantages of these methods are that the antibodies used are difficu...
View more

Abstract

The invention relates to a thrombin aptamer sensor based on Ti3C2 and a preparation method of the thrombin aptamer sensor which belong to the technical field of biological analysis and detection, andsolve the technical problems that an antibody used in a traditional thrombin detection method is difficult to prepare and store, and the related reactions are complex and time-consuming. The thrombinaptamer sensor based on Ti3C2 is composed of a single layer of Ti3C2 and a fluorescein-labeled thrombin aptamer combined to the surface of the Ti3C2, and the sequence of the fluorescein labeled thrombin aptamer is 5 '-FAM-GGT TGG TGT TGT TGG-3'. According to the invention, the thrombin sensor is constructed by adopting a Ti3C2 material, so that the application of Ti3C2 in the biological field is broadened, and an excellent nano detection platform is constructed; the thrombin aptamer is used as a thrombin recognition agent, and compared with a traditional antibody, the thrombin aptamer is low in price, is easy and convenient to synthesize, is easy to store and is rapid in reaction. The Ti3C2-based thrombin aptamer sensor prepared by the preparation method provided by the invention has the characteristics of high specificity and high sensitivity, and the detection limit of thrombin is 2.5*10-11 mol/L.

Application Domain

Fluorescence/phosphorescence

Technology Topic

AntibodyFluorescein +5

Image

  • Thrombin aptamer sensor based on Ti3C2 and preparation method of thrombin aptamer sensor
  • Thrombin aptamer sensor based on Ti3C2 and preparation method of thrombin aptamer sensor
  • Thrombin aptamer sensor based on Ti3C2 and preparation method of thrombin aptamer sensor

Examples

  • Experimental program(4)

Example Embodiment

[0039] The invention also provides a Ti-based 3 C 2 The preparation method of thrombin aptamer sensor includes the following steps:
[0040] (1) Single Ti 3 C 2 Preparation:
[0041] Ti is prepared by sintering a mixture of titanium powder, aluminum powder and graphite under argon 3 AlC 2 , Will Ti 3 AlC 2 The powder is added to the LiF/HCl solution for etching under continuous stirring. After the reaction is completed, the acid mixture is washed with ultrapure water by repeated centrifugation until the pH value of the mixture exceeds 5. Finally, the slurry is sonicated and centrifuged to obtain a monolayer Ti 3 C 2 Solution
[0042] (2) Binding of fluorescein-labeled thrombin aptamer to Ti 3 C 2 surface:
[0043] Add the fluorescein-labeled thrombin aptamer to the Ti prepared in step (1) at room temperature 3 C 2 In solution, after the reaction is complete, the composition is based on Ti 3 C 2 Thrombin aptamer sensor.
[0044] Preferably, the ratio of titanium powder: aluminum powder: graphite substance in step (1) is 5:2:3.
[0045] Preferably, the sintering temperature in step (1) is 1650° C. and the time is 2 hours.
[0046] Preferably, in step (1) Ti 3 AlC 2 The powder should be ground in a mortar and etched after passing through a 400 mesh screen.
[0047] Preferably, in step (1) Ti 3 AlC 2 The steps of powder etching are as follows:
[0048] 1g Ti after sieving 3 AlC 2 It was added to 8-12M LiF/6-9M HCl solution for etching, and the reaction was carried out at room temperature for 24 hours.
[0049] Preferably, after the etching in step (1) is completed, the centrifugation is repeated, the mixture is washed with ultrapure water, and the rotation speed is 8000 rpm every 5 minutes until the pH value of the mixture exceeds 5. Finally, the slurry is ultrasonicated for more than 10 minutes, and Centrifuge at 3500rpm for 1h.
[0050] Preferably, in step (1) Ti 3 C 2 The procedure for confirming the concentration of the solution is as follows: filter a single layer of Ti with a cellulose membrane with a pore size of 0.22μm 3 C 2 Solution, will Ti 3 C 2 After drying, the Ti 3 C 2 The film is peeled off and weighed, it can be calculated.
[0051] Preferably, the specific steps of step (2) are: adding 20-40 nM fluorescein-labeled thrombin aptamer to the 0.1-0.2 mg/mL Ti prepared in step (1) 3 C 2 In the solution, the reaction proceeds for 5 minutes, and the composition is based on Ti 3 C 2 Thrombin aptamer sensor.
[0052] Use the Ti-based 3 C 2 The steps of thrombin aptamer sensor to detect thrombin are as follows:
[0053] (1) Draw a thrombin standard curve: prepare a thrombin solution with a known concentration gradient and add it to the Ti-based solution prepared in step (2) above. 3 C 2 In the thrombin aptamer sensor solution, react for 20-30 minutes. After centrifugation, take the supernatant and measure the fluorescence spectrum. Take the fluorescence intensity of each standard sample at a wavelength of 517 nm. The fluorescence intensity is the vertical coordinate and the thrombin concentration is the horizontal Coordinates, draw a standard curve, and get a linear equation.
[0054] (2) Measure the thrombin concentration of the test solution: take the test solution and add it to the Ti-based solution prepared in step (2) above. 3 C 2 In the thrombin aptamer sensor solution, react for 20-30 minutes, take the supernatant after centrifugation, measure the fluorescence spectrum, obtain the fluorescence intensity at the wavelength of 517nm, and compare with the standard curve to obtain the thrombin concentration of the solution to be tested.
[0055] Preferably, in step (1) of the thrombin detection process, the centrifugal speed is 9000 rpm, the centrifugal time is 10 minutes, and the excitation light wavelength of the fluorescence spectrum is 470 nm.
[0056] Preferably, in step (2) of the thrombin detection process, the centrifugal speed is 9000 rpm, the centrifugal time is 10 minutes, and the excitation light wavelength of the fluorescence spectrum is 470 nm.
[0057] Combine figure 1 Explain that the Ti-based 3 C 2 The principle of thrombin aptamer sensor for detecting thrombin is: the thrombin aptamer is labeled with fluorescein and binds to Ti 3 C 2 On the surface, due to the resonance transfer of fluorescence energy, the fluorescence is 3 C 2 Effectively quench. In the presence of thrombin, due to the high affinity between thrombin and aptamer, Ti 3 C 2 The aptamer on the surface will bind to thrombin to form a quadruplex. This process causes fluorescein from Ti 3 C 2 The surface is detached, the fluorescence energy resonance transfer efficiency is reduced, and fluorescence recovery is observed. Moreover, the degree of change in the fluorescence spectrum reflects the amount of thrombin in the solution.

Example Embodiment

[0061] Example 1:
[0062] 1) Single Ti 3 C 2 Synthesis: Ti is prepared by sintering a mixture of 7.368g titanium powder, 1.523g aluminum powder and 1.109g graphite at 1650℃ under argon atmosphere, and sintering for 2 hours 3 AlC 2. Fired Ti 3 AlC 2 The powder was ground in a mortar and passed through a 400 mesh screen. 1gTi 3 AlC 2 Add to 12M LiF/9M HCl solution for etching under continuous stirring, and react at room temperature for 24 hours. After the etching is completed, the centrifugation is repeated, and the mixture is washed with ultrapure water, and the rotation speed is 8000 rpm every 5 minutes, until the pH value of the mixture exceeds 5. Finally, the slurry was sonicated for more than 10 minutes, and centrifuged at 3500 rpm for 1 hour to obtain a monolayer of Ti 3 C 2 Solution. To confirm Ti 3 C 2 The concentration of 10mL Ti is filtered with a cellulose membrane with a pore size of 0.22μm 3 C 2 Dispersions. Will Ti 3 C 2 After drying, the Ti 3 C 2 The film is peeled off and weighed, and Ti is calculated 3 C 2 concentration. figure 2 Ti 3 C 2 Scanning Electron Microscope image. image 3 Ti 3 AlC 2 And Ti 3 C 2 XRD pattern.
[0063] 2) Preparation of thrombin aptamer sensor: 40nM fluorescein-labeled thrombin aptamer was added to 0.2mg/mLTi 3 C 2 In the solution, react for 5 minutes to form a thrombin aptamer sensor.
[0064] 3) Drawing of thrombin standard curve: prepare 8 kinds of thrombin standard solutions with different concentrations, the concentrations are 25pmol/L, 50pmol/L, 75pmol/L, 100pmol/L, 125pmol/L, 150pmol/L, 175pmol/L and 200pmol/L. Add the thrombin solution to the thrombin sensor in 2), react at room temperature for 20 minutes, centrifuge at 9000 rpm, centrifuge for 10 minutes, and take the supernatant. Use Shimadzu RF-5301PC fluorescence spectrometer to detect the fluorescence spectrum. The excitation light wavelength is 470 nm. The fluorescence intensity of a standard sample at a wavelength of 517 nm, the fluorescence intensity as the ordinate, and the thrombin concentration as the abscissa, draw a standard curve to obtain a linear equation. Figure 4 It is the fluorescence spectrum image of thrombin solution of different concentration added to the thrombin sensor. Figure 5 Is the thrombin standard curve obtained.
[0065] 4) Detect the thrombin in the sample: take the sample solution and add it to the thrombin sensor in 2), react at room temperature for 20 minutes, centrifuge at 9000 rpm, centrifuge for 10 minutes, take the supernatant, and use Shimadzu RF-5301PC fluorescence spectrometer to detect fluorescence Spectrum, excitation light wavelength 470nm, take the fluorescence intensity at 517nm wavelength of the sample, and calculate the thrombin concentration in the sample according to the thrombin standard curve.

Example Embodiment

[0066] Example 2:
[0067] 1) Single Ti 3 C 2 Synthesis of Ti: Ti was prepared by sintering a mixture of 3.684g titanium powder, 0.762g aluminum powder and 0.555g graphite at 1650℃ under argon atmosphere, and sintering for 2 hours. 3 AlC 2. Fired Ti 3 AlC 2 The powder was ground in a mortar and passed through a 400 mesh screen. 1gTi 3 AlC 2 Add to 8M LiF/6M HCl solution for etching under continuous stirring, and react at room temperature for 24 hours. After the etching is completed, the centrifugation is repeated, and the mixture is washed with ultrapure water, and the rotation speed is 8000 rpm every 5 minutes, until the pH value of the mixture exceeds 5. Finally, the slurry was sonicated for more than 10 minutes, and centrifuged at 3500 rpm for 1 hour to obtain a monolayer of Ti 3 C 2 Solution. To confirm Ti 3 C 2 The concentration of 10mL Ti is filtered with a cellulose membrane with a pore size of 0.22μm 3 C 2 Dispersions. Will Ti 3 C 2 After drying, the Ti 3 C 2 The film is peeled off and weighed, and Ti is calculated 3 C 2 concentration.
[0068] 2) Preparation of thrombin aptamer sensor: 20nM fluorescein-labeled thrombin aptamer was added to 0.1mg/mLTi 3 C 2 In the solution, react for 5 minutes to form a thrombin aptamer sensor.
[0069] 3) Drawing of thrombin standard curve: prepare 8 kinds of thrombin standard solutions with different concentrations, the concentrations are 25pmol/L, 50pmol/L, 75pmol/L, 100pmol/L, 125pmol/L, 150pmol/L, 175pmol/L and 200pmol/L. Add the thrombin solution to the thrombin sensor in 2), react at room temperature for 20 minutes, centrifuge at 9000 rpm, centrifuge for 10 minutes, and take the supernatant. Use Shimadzu RF-5301PC fluorescence spectrometer to detect the fluorescence spectrum. The excitation light wavelength is 470 nm. The fluorescence intensity of a standard sample at a wavelength of 517 nm, the fluorescence intensity as the ordinate, and the thrombin concentration as the abscissa, draw a standard curve to obtain a linear equation.
[0070] 4) Detection of thrombin in the sample: Take the sample solution and add it to the thrombin sensor in 2), react at room temperature for 25 minutes, centrifuge at 9000 rpm, centrifuge for 10 minutes, take the supernatant, and use Shimadzu RF-5301PC fluorescence spectrometer to detect fluorescence Spectroscopy, the excitation light wavelength is 470nm, the fluorescence intensity at the wavelength of 517nm of the sample is taken, and the thrombin concentration in the sample is calculated according to the thrombin standard curve.

PUM

no PUM

Description & Claims & Application Information

We can also present the details of the Description, Claims and Application information to help users get a comprehensive understanding of the technical details of the patent, such as background art, summary of invention, brief description of drawings, description of embodiments, and other original content. On the other hand, users can also determine the specific scope of protection of the technology through the list of claims; as well as understand the changes in the life cycle of the technology with the presentation of the patent timeline. Login to view more.

Similar technology patents

Method for preparing composite electrode material of super capacitor

InactiveCN102903531Alow priceThe operation process is simple
Owner:DONGHUA UNIV

Antistatic coating

InactiveCN102690582Alow priceSimple construction
Owner:TIANCHANG JULONG TRAVEL PAINT

Method of manufacturing ink jet recording head

InactiveUS20070178248A1high accuracylow price
Owner:CANON KK

A mobile phone and a power saving method for a mobile phone in a sleep mode

InactiveCN102300298Alow priceSimplify hardware and software design
Owner:HUIZHOU TCL MOBILE COMM CO LTD

External traditional Chinese medicine for treating skin diseases

ActiveCN103341104Alow priceExtensive sources of raw materials
Owner:李光辉

Classification and recommendation of technical efficacy words

  • Easy to synthesize
  • low price
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents.

© 2023 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap