Fluorescent biosensor for detecting N-terminal B-type pro-natriuretic peptide as well as preparation method and application of fluorescent biosensor

Abstract

The invention provides a fluorescent biosensor for detecting N-terminal B-type pro-natriuretic peptide as well as a preparation method and application of the fluorescent biosensor. The fluorescent biosensor is mainly composed of a palladium nanosheet and a fluorescent dye modified nucleic acid aptamer of N-terminal B-type pro-natriuretic peptide. Fluorescence resonance energy transfer and an inner filtering effect can be generated between the palladium nanosheet and the fluorescent dye modified nucleic acid aptamer of the N-terminal B-type pro-natriuretic peptide, so that fluorescence of a system is quenched, a fluorescence signal is closed, and the fluorescent dye modified nucleic acid aptamer can be used for specifically recognizing the N-terminal B-type pro-natriuretic peptide, so that the sensitivity of the system is greatly improved. The fluorescent dye modified nucleic acid aptamer is far away from the palladium nanosheet, the system fluorescence signal is open, the fluorescence recovery efficiency and the logarithm of the N-terminal B-type pronatriuretic peptide have a good linear relationship in the concentration range of 0.8-30 pg / mL, and the detection limit is 0.21 pg / mL. The sensitive, rapid and specific detection of the N-terminal B-type pro-natriuretic peptide in a short time is realized through the change of the fluorescence intensity of the system.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and biochemistry, and in particular to a fluorescent biosensor for detecting N-terminal pro-B-type natriuretic peptide, and a preparation method and application thereof. Background Art

[0002] Heart failure (HF) is one of the most common cardiovascular diseases and is defined as symptoms or signs caused by abnormalities in cardiac structure or function. In recent years, its 5-year mortality rate is about 50%. N-Terminal Pro-Brain Natriuretic Peptide (NT-proBNP) is one of the chemical structures after proBNP degradation. It is widely used as a biomarker for heart failure (mainly ventricular dysfunction), mainly for the diagnosis and prognosis of heart failure.

[0003] At present, a variety of immunoassay methods have been applied to the specific detection of NT-proBNP, including fluorescence, electrochemical, chemiluminescent, etc., but most of them use antibodies as recognition elements. Since the 3D structure of antibodies may be unstable during in vitro labeling, the cost is high and it is not easy to promote. Therefore, the development of a rapid, sensitive, highly selective and low-cost method for detecting NT-proBNP is of great significance for the early diagnosis and prognosis of heart failure.

[0004] Aptamers are short single-stranded RNA or DNA-based oligonucleotides that can bind to a variety of ligands through non-covalent interactions and have the advantages of being easy to chemically modify and chemically stable. Compared with antibodies, detection methods using aptamers as recognition elements have more advantages. Palladium Nanosheets (Pd NSs) are a type of precious metal nanomaterial with a two-dimensional structure. They have good absorption properties in the near-infrared region, a large specific surface area, and size adjustability. The above characteristics lay the foundation for its widespread application in the biomedical field.

[0005] In recent years, the detection method of fluorescent biosensors has attracted much attention in the biomedical field due to its advantages of simplicity, low cost, high sensitivity, etc. Among them, the fluorescence resonance energy transfer mechanism can effectively avoid the interference of background signals, greatly improve the detection sensitivity, and promote its wide application in the fields of biology, pharmacy, and medicine. The present invention constructs a biosensor based on the fluorescence resonance energy transfer / inner filter effect dual mechanism quenching fluorescence based on the characteristics that the palladium nanosheet has good absorption characteristics in the near-infrared region and overlaps with the absorption spectrum and emission spectrum of the nucleic acid aptamer (Cy5.5-apt) of NT-proBNP modified with the fluorescent dye Cy5.5, and realizes the rapid, sensitive and specific detection of NT-proBNP. Summary of the invention

[0006] The present invention aims to provide a fluorescent biosensor for detecting NT-proBNP, a preparation method thereof, and apply the same to the detection of NT-proBNP in actual plasma samples, which has the advantages of being rapid, sensitive and highly selective.

[0007] The specific steps of the solution provided by the present invention are:

[0008] In a first aspect, the present invention provides an "off-on" type fluorescent biosensor for detecting NT-proBNP, characterized in that the fluorescent biosensor comprises a composite nanoprobe composed of palladium nanomaterials and Cy5.5-apt.

[0009] Wherein, the nucleic acid aptamer sequence is: 5'-CAG GGG GAC GGG TCG GGT TAC AGC TGAGTGTGG CC-3'.

[0010] In a second aspect, the present invention provides a method for preparing the fluorescent biosensor according to claim 1, comprising the following steps:

[0011] (1) PdNSs solution was prepared by chemical synthesis using palladium acetylacetonate as raw material.

[0012] (2) The PdNSs solution and Cy5.5-apt were vortexed to mix well to obtain a composite nanoprobe of PdNSs and Cy5.5-apt.

[0013] In a third aspect, the present invention also provides a method for detecting NT-proBNP, comprising the following steps: vortex mixing the above-mentioned composite nanoprobe with standard solutions of NT-proBNP of different concentrations (concentrations are 0.80, 1.0, 1.5, 3.0, 8.0, 10, 12, 15, 20, 25, and 30 pg / mL), and plotting its fluorescence spectrum in the range of 690 nm-750 nm after standing in the dark, and according to the change of its fluorescence intensity at the maximum emission wavelength of 675 nm, its fluorescence recovery efficiency is linearly related to the logarithm of the NT-proBNP concentration within a specific range, thereby establishing a corresponding standard curve.

[0014] Wherein, in the fluorescent biosensor, the concentration of PdNSs is 20-60 μg / mL, the pH of the reaction system is 6.5-8.5, the reaction time is 0-6 min, and the incubation time is 0-20 min.

[0015] The fluorescence recovery efficiency of the fluorescence sensor is linearly correlated within the range of NT-proBNP concentration of 0.8 to 30 pg / mL.

[0016] As described above, the present invention has the following beneficial effects:

[0017] 1. The present invention constructs a fluorescent biosensor, which utilizes the dual mechanism of fluorescence resonance energy transfer / inner filter effect to quench fluorescence, thereby greatly improving the fluorescence quenching efficiency, increasing the sensitivity of detecting NT-proBNP, and being able to achieve specific detection of NT-proBNP in a short time.

[0018] 2. The present invention uses nucleic acid aptamers as recognition elements, which can avoid cross-reactions between antibodies and antigens, does not require magnetic bead separation, is low-cost, and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The composite nanoprobe of the present invention is a fluorescence resonance energy transfer / inner filter effect dual mechanism fluorescent biosensor for detecting NT-proBNP;

[0020] Figure 2 Transmission electron microscopy image of PdNSs;

[0021] Figure 3 To verify the feasibility of this method in detecting NT-proBNP;

[0022] Figure 4 Visible-near infrared absorption spectra of PdNSs and absorption and emission spectra of Cy5.5-apt;

[0023] Figure 5 It is the fluorescence intensity change spectrum of the fluorescence recovery of the system with the change of NT-proBNP concentration;

[0024] Figure 6 It is the standard curve corresponding to the fluorescence recovery efficiency of the system and the logarithm of the NT-proBNP concentration;

[0025] Figure 7 This is a comparison chart of the fluorescence recovery efficiency of the system with different substances added;

[0026] Figure 8 To explore the anti-interference ability of this method for NT-proBNP detection (Group 1 is IL-6, DD, proBNP, BSA, Hb, Gly, CNP; Group 2 is a mixed system with the same concentration of NT-proBNP added to Group 1, and Group 3 is pure NT-proBNP. The concentrations of NT-proBNP and other interfering substances in the system are both 3.0 pg / mL). DETAILED DESCRIPTION

[0027] The following is intended to better illustrate the present invention in combination with specific embodiments and diagrams.

[0028] Example 1 Synthesis of Palladium Nanomaterials

[0029] (1) 32 mg of palladium acetylacetonate, 180 mg of citric acid, 120 mg of hexadecyltrimethylammonium bromide, and 60 mg of polyvinylpyrrolidone were placed in a round-bottom flask, and 20 mL of N,N-dimethylformamide was added. After stirring for 1 hour, a reaction solution was obtained, which was orange-red. The rotation speed was 600 r / min. 200 mg of hexacarbonyl tungsten was added while passing argon gas, and the reaction was continued by stirring for 1 hour. The reaction solution was dark green. Reaction conditions: Argon flow rate was 200 mL min -1 , the reaction temperature is 80℃.

[0030] (2) After cooling, centrifugation and washing were repeated three times to obtain precipitated PdNSs, and the washing solvent used was acetone. The precipitate was dissolved in a 20 mL PdNSs solution, and the solvent was anhydrous ethanol, and stored at 4°C.

[0031] The transmission electron microscopy results of the prepared Pd NSs are shown in Figure 2 As shown in Figure 2, the prepared Pd NSs have a uniformly dispersed hexagonal structure with a side length of 6.98±0.16nm. Figure 3 It can be seen that it has a very broad absorption peak in the range of 600-1100nm.

[0032] Example 2 Preparation of composite nanoprobes

[0033] (1) Add 20 μL of PdNSs dispersion (800 μg / mL), 100 μL of Tris-HCl buffer (pH 7.4), and 20 μL of Cy5.5-apt solution (100 nM) into an EP tube in sequence and vortex to mix.

[0034] (2) After standing at room temperature for 3 min, the PdNSs-Cy5.5-apt composite nanoprobe was obtained.

[0035] Example 3 Feasibility verification of this method for detecting NT-proBNP

[0036] (1) Six control groups (Cy5.5-apt, Cy5.5-apt+NT-proBNP, Cy5.5-apt+PdNSs, NT-proBNP+Pd NSs, NT-proBNP, Pd NSs) and an experimental group (Cy5.5-apt+Pd NSs+NT-proBNP) were set up for experiments.

[0037] (2) Prepare the composite nanoprobe according to Example 2.

[0038] (3) Add 30 μL NT-proBNP (40 pg / mL) and finally add ultrapure water to make up the total volume to 400 μL. Incubate in a dark place for 10 min and measure the fluorescence absorbance.

[0039] from Figure 3 It can be seen that when PdNSs exist in the system, the fluorescence of the system is effectively quenched, with a quenching efficiency of up to 81.5%. After the addition of NT-proBNP, due to the high affinity between Cy5.5-apt and NT-proBNP, the fluorescence of the system is restored after the two combine. The results show that it is feasible to detect NT-proBNP using this method.

[0040] Example 4 Sensitive Detection of NT-proBNP

[0041] (1) Eleven groups of NT-proBNP with different concentrations were set as experimental groups, from low to high: 0.8, 1.0, 1.5, 3.0, 8.0, 10, 12, 15, 20, 25, and 30 pg / mL.

[0042] (2) Prepare the composite nanoprobe according to Example 2.

[0043] (3) Add different concentrations of NT-proBNP and finally add ultrapure water to make up the total volume to 400 μL. After standing in the dark for 10 min, measure the fluorescence absorbance.

[0044] from Figure 5 , Figure 6 It can be seen that the change in the fluorescence intensity of the system is positively correlated with the change in the NT-proBNP concentration, and is linearly correlated when the NT-proBNP concentration is in the range of 0.80-30 pg / mL. The resulting linear regression equation is: (F2-F1) / (F0-F1)=0.238lnc+0.139(r=0.998), and the calculated detection limit is 0.21 pg / mL, indicating that this method has a high sensitivity in detecting NT-proBNP.

[0045] Example 5 Specific detection of NT-proBNP

[0046] (1) The same concentration of NT-proBNP (3.0 pg / mL) and other 21 groups of different interfering substances (3.0 pg / mL) were set as the control group.

[0047] (2) Prepare the composite nanoprobe according to Example 2.

[0048] (3) Add the same concentration of NT-proBNP and interfering substances, and finally add ultrapure water to make up the total volume to 400 μL. After standing for 10 min, measure the fluorescence absorption intensity.

[0049] from Figure 7 It can be seen that the fluorescence recovery efficiency of the group with NT-proBNP added to the system is relatively large, greater than 40%. In contrast, the fluorescence recovery efficiency of the system with other interfering substances is relatively low, all below 10%, which is less than 1 / 4 of the recovery efficiency of the NT-proBNP group, indicating that this method can specifically react with NT-proBNP and has good selectivity.

[0050] Example 6 Test of the anti-interference ability of the method for detecting NT-proBNP

[0051] (1) Three groups of solutions, Group 1, Group 2 and Group 3, were set up for the experiment. Group 1 contained IL-6, DD, proBNP, BSA, Hb, Gly and CNP. Group 2 contained the same concentration of NT-proBNP as Group 1. Group 3 contained only NT-proBNP. The concentrations of NT-proBNP and other interfering substances in the system were both 3.0 pg / mL.

[0052] (2) Prepare the composite nanoprobe according to Example 2.

[0053] (3) Add the same concentration of NT-proBNP and interfering substances (both at 3.0 pg / mL), and finally add ultrapure water to make up the total volume to 400 μL. After standing for 10 min, measure the fluorescence absorption intensity.

[0054] from Figure 8 It can be seen that the fluorescence recovery efficiency of the mixed solution group with NT-proBNP and other interfering substances is equivalent to that of the simple NT-proBNP group, indicating that the presence of external interfering substances has no obvious effect on the fluorescence signal response of NT-proBNP by this method. Therefore, this method has good anti-interference ability for detecting NT-proBNP.

[0055] Example 6 This method is used to detect NT-proBNP in actual plasma samples

[0056] Three groups of NT-proBNP with different spiked concentrations, namely low (1 pg / mL), medium (12 pg / mL), and high (29 pg / mL), were set up for the experiment. Each group was run three times in parallel. The fluorescence recovery efficiency and spiked recovery rate were calculated based on the changes in the fluorescence intensity of the system.

[0057]

[0058] Table 1. Results of spiked test using this method in actual plasma samples

[0059]

[0060] The experimental results are shown in Table 1. The spiked recovery rate of NT-proBNP in plasma by this method is 99.95% to 120.79%, and the relative standard deviation obtained by three parallel experiments is 2.90% to 6.85% (n=3), indicating that this method has good accuracy in the detection of NT-proBNP in actual plasma samples.

[0061] The above experimental results describe the experimental principle of the present method and its advantages for detecting NT-proBNP. The above embodiments are not intended to limit the patent protection scope of the present invention. On this basis, the present invention will be continuously optimized.

Claims

1. An "off-on" fluorescent biosensor for detecting N-terminal pro-B-type natriuretic peptide, characterized in that: The fluorescent biosensor comprises a palladium nanosheet and a composite nanoprobe composed of a nucleic acid aptamer of an N-terminal pro-B-type natriuretic peptide modified with a fluorescent dye.

2. The fluorescent biosensor according to claim 1, characterized in that: The sequence of the N-terminal pro-B-type natriuretic peptide nucleic acid aptamer is: 5'-CAG GGG GAC GGG TCG GGT TAC AGC TGAGTG TGG CC-3'.

3. The method for preparing the fluorescent biosensor according to claim 1, characterized in that: The following steps are involved: (1) Using acetylacetonate palladium as raw material, palladium nanosheet solution was prepared by chemical synthesis method. (2) Vortexing the palladium nanosheet solution and the fluorescent dye-modified N-terminal pro-B-type natriuretic peptide nucleic acid aptamer to obtain a composite nanoprobe of the palladium nanosheet and the fluorescent dye-modified N-terminal pro-B-type natriuretic peptide nucleic acid aptamer.

4. A method for detecting N-terminal pro-B-type natriuretic peptide, characterized in that: The following steps are involved: The composite nanoprobe and standard solutions of N-terminal pro-B-type natriuretic peptide of different concentrations were vortexed and mixed, and then the fluorescence spectrum in the range of 690nm-750nm was plotted after standing in the dark. According to the change of fluorescence intensity at the maximum emission wavelength of 675nm, the fluorescence recovery efficiency was proportional to the exponential of the N-terminal pro-B-type natriuretic peptide concentration. The corresponding linear regression equation was constructed, and the concentration of N-terminal pro-B-type natriuretic peptide could be obtained by calculating the fluorescence recovery efficiency of the reaction solution.

5. The method for detecting N-terminal pro-B-type natriuretic peptide according to claim 4, characterized in that: The pH value of the fluorescent biosensor reaction system is 6.5-8.

5.

6. The method for detecting N-terminal pro-B-type natriuretic peptide according to claim 4, characterized in that: The reaction time of the fluorescent biosensor is 0 to 6 minutes.

7. The method for detecting N-terminal pro-B-type natriuretic peptide according to claim 4, characterized in that: The incubation time of the fluorescent biosensor is 0 to 20 minutes.

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