Fluorescent Aptasensor for Low-Density Lipoprotein Detection Based on N,S-GQDs-Fe3O4@rGO

Abstract

Based on N,S-GQDs-Fe3O4@rGO, a fluorescence aptamer sensor for the detection of low-density lipoprotein was constructed. Utilizing the good fluorescence quenching ability of the Fe3O4@reduced graphene oxide (Fe3O4@rGO) composite material and the high fluorescence intensity of nitrogen and sulfur co-doped quantum dots (N,S-GQDs), the LDL aptamer can specifically recognize and bind to LDL protein, thus constructing a novel aptamer fluorescence aptamer sensor capable of specifically and quantitatively detecting LDL protein for detecting the content of LDL in serum. This method is simple to operate, low in cost and has a low detection limit of 1.29 ng / mL.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical sensing, and particularly relates to a low-density lipoprotein fluorescent aptamer sensor based on fluorescence resonance energy transfer occurring between composite materials. Background Art

[0002] Common detection methods for low-density lipoprotein (LDL) are colloidal gold method, immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), etc., which have problems such as high price, poor applicable environment, and difficulty in being carried out by grass-roots units. The invention patent with the publication number CN201610219116.3 discloses an enzyme-linked immunosorbent assay kit for detecting oxidized low-density lipoprotein. This detection method has problems such as low judgment accuracy, easy occurrence of false positives, long detection time, and complex operation. Fluorescence resonance energy transfer (FRET) is an energy migration phenomenon that occurs between two fluorescent molecules with very close distances. The fluorescence intensity of the donor is much lower than when it exists alone (fluorescence quenching), while the fluorescence emitted by the acceptor is greatly enhanced (sensitized fluorescence). Developing a new type of low-cost fluorescence aptamer sensor based on the FRET phenomenon for LDL detection is a new development direction. The invention patent with the publication number CN111855988A discloses a method for fluorescently detecting low-density lipoprotein based on fluorescent particles combined with low-density lipoprotein antibodies. This method has problems such as weak binding ability between antibodies and low-density lipoprotein and complex operation. Therefore, developing a new type of low-cost, high-sensitivity, and highly specific fluorescence aptamer sensor is a new method for detecting LDL. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an LDL fluorescent aptamer sensor based on the fluorescence resonance energy transfer (FRET) phenomenon of nitrogen and sulfur co-doped graphene quantum dots - low-density lipoprotein aptamer - iron tetroxide @ reduced graphene oxide (N,S-GQDs-LDL Apt -Fe3O4@rGO), and utilize the high fluorescence performance of nitrogen and sulfur co-doped graphene quantum dots (N,S-GQDs), the specific binding ability of low-density lipoprotein aptamer (LDL Apt ) and the strong fluorescence quenching ability of iron tetroxide @ reduced graphene oxide (Fe3O4@rGO) to improve the detection efficiency of LDL and improve the sensitivity. This method can achieve a detection limit of 1.29 ng / mL.

[0004] To solve this technical problem: N,S-GQDs are used as fluorescent substances, and then the carboxyl groups on the surface of N,S-GQDs are activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and amino-LDLApt It is combined with N,S-GQDs through dehydration condensation via amino and carboxyl bonds to form fluorescently labeled N,S-GQDs-LDL Apt complex. In the N,S-GQDs-LDL Apt solution, add the Fe3O4@rGO dispersion. The N,S-GQDs-LDL Apt complex and Fe3O4@rGO are combined together through van der Waals forces, and fluorescence resonance energy transfer occurs, causing the fluorescence energy of N,S-GQDs-LDL Apt to transfer to Fe3O4@rGO, and the fluorescence intensity of N,S-GQDs-LDL Apt will become lower; after adding LDL protein, due to the specificity of LDL Apt , LDL will preferentially bind to N,S-GQDs-LDL Apt , the aptamer structure changes, separates from the bottom surface of Fe3O4@rGO, inhibits fluorescence resonance energy transfer, and thus restores the fluorescence of N,S-GQDs-LDL Apt . According to the change in the recovery degree of the fluorescence intensity of the fluorescence aptamer sensor, establish a linear relationship between the LDL concentration and the change in the fluorescence intensity of N,S-GQDs-LDL Apt to achieve highly sensitive and selective quantitative detection of LDL.

[0005] The present invention is carried out according to the following steps:

[0006] Step 1: Preparation of the fluorescence energy donor N,S-GQDs-LDL Apt

[0007] (1) Preparation of nitrogen and sulfur co-doped graphene quantum dots (N,S-GQDs): Prepare a solution of citric acid and thiourea in proportion, then stir and heat. After the reaction is completed, cool, centrifuge, and dry to obtain N,S-GQDs solid;

[0008] (2) Preparation of nitrogen and sulfur co-doped graphene quantum dot - low density lipoprotein aptamer (N,S-GQDs-LDL Apt ) : Add EDC to the N,S-GQDs solution to activate the carboxyl groups on the surface of N,S-GQDs. Measure LDL Apt , mix evenly with the N,S-GQDs solution in equal proportion, and stir and mix for a certain time at room temperature and in the dark to obtain the N,S-GQDs-LDL Apt solution.

[0009] Step 2: Construction of the LDL fluorescence aptamer sensor

[0010] ​(1) Weigh the reduced graphene oxide rGO, add it to N,N-dimethylformamide DMF, and use an ultrasonic cell disruptor to prepare a dispersion; add iron oxide Fe3O4 to ethanol and use an ultrasonic cell disruptor to prepare a dispersion; mix the two dispersions, heat and stir, and obtain Fe3O4@rGO powder after drying; weigh the Fe3O4@rGO powder, add ultrapure water to make up the volume, and put it into an ultrasonic cell disruptor to break it until the Fe3O4@rGO powder is completely dispersed in ultrapure water, thus obtaining the Fe3O4@rGO dispersion;

[0011] (2) Mix the Fe3O4@rGO solution and the N,S-GQDs-LDL Apt solution, incubate for a period of time after mixing evenly, quench the fluorescence of N,S-GQDs, and form an LDL fluorescent aptamer sensor based on the FRET phenomenon of N,S-GQDs-LDL Apt -Fe3O4@rGO. Scan it with a fluorescence spectrophotometer and measure its fluorescence intensity, denoted as F0.

[0012] Step 3: Plotting the LDL working curve

[0013] (1) Add LDL solutions with different concentrations to the fluorescent aptamer sensor, incubate and react at a certain temperature for a period of time, scan it with a fluorescence spectrophotometer, and measure its fluorescence intensity, denoted as F1

[0014] (2) Use (F1 - F0) / F0 as the ordinate and the LDL concentration as the abscissa to plot the working curve, and calculate the sensitivity and the lowest detection limit of this method.

[0015] Step 4: Detection of LDL in actual samples

[0016] (1) Mix the actual serum sample and the LDL standard solution to obtain the sample to be tested, add the sample to be tested to the LDL fluorescent aptamer sensor in Step 2, incubate and react at a certain temperature for a period of time, scan it with a fluorescence spectrophotometer, fix the excitation wavelength at 368 nm, and record the fluorescence intensity at 450 nm;

[0017] (2) Calculate the concentration of LDL in the sample to be tested according to the LDL working curve obtained in Step 3.

[0018] Further optimization:

[0019] The DNA sequence of LDL in Step 1 Apt is 5′-ACC TCG ATT TTA TAT TAT TTC GCT TAC CAACAA CTG CAG A-NH2-3′;

[0020] In step 1, the heating conditions are 160 °C for 8 h; under these conditions, the yield of N,S-GQDs is relatively high;

[0021] The concentration of EDC in step 1 is 1 mg / mL;

[0022] In step 1, LDL Apt is 1.5 μmol / L;

[0023] In step 1, the activation time is 40 min and the incubation time is 1 h. Under these conditions, the binding effect between N,S-GQDs and LDL Apt is relatively good;

[0024] In step 2, the concentration of the Fe3O4@rGO solution is 0.01 mg / mL;

[0025] In step 2, N,S-GQDs-LDL Apt has a concentration of 50 μg / mL;

[0026] In step 2, the volume ratio of the Fe3O4@rGO solution to the N,S-GQDs-LDL Apt solution is 2:1;

[0027] In step 2, the incubation temperature is 25 °C and the incubation time is 80 min. Under these conditions, the binding effect between N,S-GQD-LDL Apt and Fe3O4@rGO is relatively good;

[0028] In steps 2, 3, and 4, the excitation wavelength of the fluorescence spectrophotometer is 368 nm and the emission wavelength is 450 nm. Under these conditions, the fluorescence intensity is relatively high;

[0029] In steps 3 and 4, the incubation temperature is 25 °C and the incubation time is 80 min.

[0030] Among them, in step 1, a nano-fluorescent material of N,S-GQDs that emits blue fluorescence and an N,S-GQDs-LDL Apt probe are obtained, which serve as the fluorescence energy donor for the FRET phenomenon in step 2. Step 2 provides the fluorescence energy transfer acceptor Fe3O4@rGO and constructs an LDL fluorescence aptasensor; by using the nucleic acid aptamer bases in N,S-GQDs-LDL Apt and Fe3O4@rGO through van der Waals forces and π-π conjugation, N,S-GQDs-LDL Apt and Fe3O4@rGO are closely approached, resulting in the FRET phenomenon and presenting fluorescence quenching of N,S-GQDs-LDL Apt When LDL is introduced into the LDL fluorescence aptasensor in step 3, due to LDL AptPreferentially bind to LDL, thus successfully weakening the interaction between N,S-GQDs-LDL Apt and Fe3O4@rGO. Therefore, N,S-GQDs-LDL Apt is separated from Fe3O4@rGO, the FRET process is inhibited, and the fluorescence of N,S-GQDs-LDL Apt is restored. The working curve of LDL in Step 3 provides a calculation basis for the determination of LDL concentration in the actual samples in Step 4. The experimental results of Steps 1-4 prove that a novel LDL fluorescent aptamer sensor can be established by using the FRET phenomenon between N,S-GQDs-LDL Apt and Fe3O4@rGO.

[0031] The present invention has the following advantages compared with the prior art:

[0032] 1. This method makes full use of the large specific surface area and conductivity of rGO to construct a Fe3O4@rGO composite material with excellent fluorescence quenching properties, and combines the high fluorescence intensity of N,S-GQDs with LDL having high affinity Apt , and successfully prepares a low-density lipoprotein fluorescent aptamer sensor based on the N,S-GQDs and Fe3O4@rGO composite material. This sensor has the advantages of high sensitivity, large linear range, simple operation, etc., and provides a new method for the detection of LDL in serum.

[0033] 2. The affinity between the aptamer and the target is often stronger than that between the antigen and the antibody, which can improve the detection sensitivity and detection range. In addition, the aptamer is easier to be chemically labeled and modified than the antibody, and it is easy to construct a fluorescent aptamer sensor based on the principle of fluorescence resonance energy transfer. The detection process is simple to operate, can realize a one-step reaction, has a shorter detection time and lower cost;

[0034] 3. This sensor uses the LDL aptamer as the recognition probe to detect LDL, has the characteristic of small background interference, and can effectively improve the detection accuracy. This sensor has good specificity, stability and reproducibility, can specifically detect the LDL level in serum, and the lowest detection limit is 1.29 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the LDL fluorescent aptamer sensor for detecting LDL based on N,S-GQDs-LDL Apt -Fe3O4@rGO;

[0036] Figure 2 Fluorescence spectra of N,S-GQDs and N,S-GQDs-LDL Apt ;

[0037] Figure 3 A is the TEM image of N,S-GQDs; B is the SEM image of Fe3O4@rGO;

[0038] Figure 4 Fluorescence recovery intensity diagram of the LDL fluorescence aptamer sensor at different LDL concentrations. Specific embodiments

[0039] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] A fluorescence aptamer sensor for detecting LDL based on the FRET phenomenon between N,S-GQDs and Fe3O4@rGO Apt The detection principle of the LDL fluorescence aptamer sensor is shown in Figure 1 . First, N,S-GQDs are prepared as a fluorescent group, and LDL Apt is combined with N,S-GQDs to form fluorescently labeled N,S-GQDs-LDL Apt ; then Fe3O4@rGO is added to form the LDL fluorescence aptamer sensor, and at this time, fluorescence quenching can be observed; after adding the target LDL protein, LDL Apt specifically binds to the LDL protein, so that N,S-GQDs-LDL Apt detaches from Fe3O4@rGO, and the fluorescence of N,S-GQDs-LDL Apt is restored. Subsequently, the fluorescence intensity F1 of the combined NGQDs-LDL Apt is measured and recorded, and the LDL concentration is calculated, thus constructing a fluorescence aptamer sensor for detecting the LDL protein concentration. By measuring the change in fluorescence intensity with a fluorescence spectrophotometer, quantitative analysis of the LDL protein can be effectively achieved.

[0041] The implementation steps are as follows:

[0042] (1) Preparation of N,S-GQDs-LDL Apt Preparation

[0043] ① Dissolve 2.1 g (1 moL) of citric acid and 2.3 g (3 moL) of thiourea in 10 mL of pure water in a ratio of 1:3, place it in a water bath at 60 °C and heat and stir until it becomes a transparent solution.

[0044] ② Then transfer the transparent solution to a 20 mL high-pressure reactor, heat it to 160 °C and keep it for 8 h, and then cool it to room temperature. After that, add absolute ethanol to the solution, and then put it into a high-speed refrigerated centrifuge and centrifuge at a speed of 6000 r / min for 5 min. Then extract the supernatant to separate it from the precipitate, repeat this process several times until the precipitate is complete, and dry the centrifuged material to obtain N,S-GQDs.

[0045] ③ Measure 50 μL of the N,S-GQDs solution with a concentration of 1 μg / mL after being activated by EDC and mix it with LDL with a concentration of 1.5 μmol / L Apt in equal proportion, stir for 1 h under the conditions of room temperature and light avoidance, and then obtain the N,S-GQDs-LDL Apt solution. Figure 2 are the fluorescence spectra of N,S-GQDs and N,S-GQDs-LDL Apt , and their fluorescence spectra are basically the same. The fluorescence intensity of N,S-GQDs-LDL Apt is smaller than that of N,S-GQDs, indicating that N,S-GQDs and LDL Apt have been successfully connected.

[0046] (2) Construction of the LDL fluorescence aptasensor

[0047] ① Ultrasonically crush and disperse 1.0 g of prepared iron oxide nanoparticles into 50 mL of ethanol, and ultrasonically disperse 0.2 g of rGO into 50 mL of N,N-dimethylformamide (DMF) solvent. Mix the two dispersions, heat to 60 °C, and place it on a magnetic stirrer and stir for 2 h. Finally, transfer the mixed solution to a vacuum rotary evaporator to remove ethanol and DMF in the system. Dry the obtained mixture in an 80 °C drying oven for 5 h to obtain Fe3O4@rGO black powder.

[0048] ② Figure 3 Figure B is the scanning electron microscope image of Fe3O4@rGO. The spherical particles in the figure are Fe3O4@rGO, and the particle size is about 50 nm.

[0049] ③ Extract 100 μL of the Fe3O4@rGO dispersion with a concentration of 0.01 mg / mL and 50 μL of the N,S-GQDs-LDL Apt solution and mix them, add them to 810 μL of HEPES buffer solution with a pH of 7.0, shake and mix evenly, and incubate at 25 °C for 80 min to obtain the LDL fluorescence aptasensor. Scan with a fluorescence spectrophotometer, fix the excitation wavelength at 368 nm, and measure its fluorescence intensity at 450 nm, denoted as F0.

[0050] (3) Plotting of the LDL working curve

[0051] The LDL fluorescent aptamer sensors after measuring the fluorescence intensity in Step 2 were evenly divided into 6 groups, and then 200 μL of LDL protein solutions with concentration gradients (0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1000 ng / mL, 10000 ng / mL) were added. After shaking and mixing evenly, the reaction was carried out at 25 °C for 80 min. Scanning was performed with a fluorescence spectrophotometer. The excitation wavelength was fixed at 368 nm, and the fluorescence intensity at 450 nm was measured and denoted as F1. The fluorescence spectra of the LDL fluorescent aptamer sensors at different LDL concentrations are shown in Figure 4 , and it can be seen that the fluorescence recovery intensity ((F1 - F0) / F0) of the LDL fluorescent aptamer sensor is positively correlated with the LDL concentration. When the LDL protein concentration range is 0.1 ng / mL - 10000 ng / mL, the relationship between the fluorescence recovery value of the LDL fluorescent aptamer sensor and the LDL concentration is linear. The working curve is Y = 0.09362 + 0.01696 lgX (where y is the fluorescence recovery intensity value and x is the LDL concentration), and its correlation coefficient R 2 = 0.99219. The lowest detection limit of this LDL fluorescent aptamer sensor is 1.29 ng / mL.

[0052] (4) Detection of LDL in actual serum samples

[0053] LDL standard solutions with concentrations of 0.2 μg / mL, 2.0 μg / mL, and 20 μg / mL were fully mixed with normal human serum samples at a ratio of 1:1 to prepare a mixed solution. Then, the prepared N,S-GQDs-LDL Apt -Fe3O4@rGO fluorescent aptamer sensor was used to detect the fluorescence recovery intensity value of the mixed solution. According to the working curve y = 0.09362 + 0.01696 lgx obtained in Step 3, the corresponding concentration of LDL in the actual serum sample can be calculated. The detection results are shown in Table 1. It can be concluded that the recovery rate of the N,S-GQDs-LDL Apt -Fe3O4@rGO fluorescent aptamer sensor is 98.0% - 110%, and the relative standard deviation is between 0.39% - 0.89%. It has potential applications in the clinical diagnosis field of liver cancer.

[0054] Table 1 Detection results of LDL in actual serum samples

[0055]

Claims

1. An LDL detection method based on fluorescence resonance energy transfer of N, S-GQDs-LDL Apt -Fe3O4@rGO is carried out according to the following steps: Step 1: Preparation of Fluorescent Energy Donor N,S-GQDs-LDL Apt Preparation (1) Preparation of nitrogen and sulfur co-doped graphene quantum dots N,S-GQDs: Prepare a solution of citric acid and thiourea in a certain proportion, stir and heat. After the reaction is completed, cool, centrifuge, and dry to obtain the N,S-GQDs solid; (2) Add EDC to the N,S-GQDs solution to activate the carboxyl groups on the surface of N,S-GQDs; measure LDL Apt , mix it evenly with the N,S-GQDs solution in equal proportion, and stir and mix for a certain time under the conditions of room temperature and light avoidance to obtain the N,S-GQDs-LDL Apt solution; Step 2: Construction of the fluorescence aptasensor (1) Weigh reduced graphene oxide rGO, add it to N,N-dimethylformamide DMF, and use an ultrasonic cell disruptor to make a dispersion; add iron oxide Fe3O4 to ethanol and use an ultrasonic cell disruptor to make a dispersion; mix the two dispersions, heat and stir, and dry to obtain Fe3O4@rGO powder; weigh the Fe3O4@rGO powder, add ultrapure water to make up the volume, and put it into an ultrasonic cell disruptor to break it until the Fe3O4@rGO powder is completely dispersed in ultrapure water to obtain the Fe3O4@rGO dispersion; (2) Mix the Fe3O4@rGO solution and the N,S-GQDs-LDL Apt solution, incubate it after mixing evenly and standing still to quench the fluorescence of N,S-GQDs, and form an LDL fluorescence aptasensor; scan it with a fluorescence spectrophotometer, fix the excitation wavelength at 368 nm, and measure the fluorescence intensity F0 at 450 nm; Step 3: Plotting of the LDL working curve (1) Add LDL solutions with different concentrations to the LDL fluorescence aptasensor and incubate; perform a scan with a fluorescence spectrophotometer, fix the excitation wavelength at 368 nm, and measure the fluorescence intensity at 450 nm, denoted as F1; (2) Use (F1 - F0) / F0 as the ordinate and the LDL concentration as the abscissa to plot the working curve and calculate the lowest detection limit of this method; Step 4: Detection of LDL in actual serum samples (1) Mix the actual serum sample and the LDL standard solution to obtain the sample to be tested, add the sample to be tested to the LDL fluorescence aptasensor in Step 2, incubate, and perform a scan with a fluorescence spectrophotometer, fix the excitation wavelength at 368 nm, and record the fluorescence intensity at 450 nm; (2) Calculate the concentration of LDL in the sample to be tested according to the LDL working curve obtained in Step 3.

2. The method according to claim 1, wherein: The incubation temperature in Step 2 is 25 °C and the incubation time is 80 min.

3. The method according to claim 1, characterized in that: N,S-GQDs-LDL in Step 2 Apt The concentration is 50 μg / mL.

4. The method according to claim 1, characterized in that: The incubation temperature in Steps 3 and 4 is 25 °C and the incubation time is 80 min.

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