Lateral chromatography paper and sensor for detecting heart failure biomarker NT-proBNP

Abstract

The invention relates to the field of biological detection, and particularly discloses a lateral chromatography paper-based sensor for detecting a heart failure biomarker NT-proBNP. The lateral chromatography paper is composed of a bottom plate, and a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially overlapped and pasted on the bottom plate along a sample chromatography direction, the combination pad is coated with a gold nanoparticle coupling aptamer 1, and the aptamer 1 is an aptamer capable of being specifically combined with NT-proBNP; different positions of the detection pad are coated with NT-proBNP specific antibodies and aptamers 2, so that a detection line and a quality control line are respectively formed. The lateral chromatography paper can realize qualitative detection of heart failure biomarkers NT-proBNP in samples such as serum, plasma and urine by naked eyes, can realize quantitative detection of NT-proBNP based on application of intelligent equipment, is a simple, rapid and sensitive detection mode, and has a wide application prospect.

Description

Technical Field

[0003] The present application relates to the field of biological detection, and more specifically, to a lateral flow paper-based sensor for detecting NT-proBNP, a biomarker of heart failure. Background Art

[0004] Cardiovascular disease is the most common cause of death worldwide, and heart failure is one of the most prevalent and lethal cardiovascular diseases. The World Health Organization estimates that the number of people with heart failure is expected to increase by 46% by 2030, to 1.57 million by 2034, and the cost of cardiovascular disease medical care will exceed $1 trillion in 2035.

[0005] NT-proBNP (N-terminal pro-brain natriuretic peptide) is an inactive prohormone. As an important biomarker for heart failure, its detection is of great significance for the diagnosis, disease assessment and prognosis of heart failure. Generally, the NT-proBNP level in patients with heart failure is higher than that in patients with normal heart function. The NT-proBNP level increases significantly with the worsening of heart failure, which can reflect the severity of impaired heart function, help to identify heart failure patients early, and monitor the progression of the disease.

[0006] NT-proBNP testing has become a routine means of clinical management of heart failure and is widely used in departments such as emergency departments and cardiology departments. However, the current detection of NT-proBNP is mainly carried out through blood samples, and its concentration is quantitatively measured in the laboratory using immunological methods such as electrochemiluminescence, immunofluorescence or immunoturbidimetry. This examination is complex, time-consuming, and requires precise laboratory instrumentation, which greatly limits its wide clinical application.

[0007] In view of this, there is an urgent need to propose a method that can conveniently and quickly detect NT-proBNP, a biomarker of heart failure. Summary of the invention

[0008] In order to solve the above technical problems, the present application provides a lateral flow paper and a sensor for detecting NT-proBNP, a biomarker of heart failure. The lateral flow paper can realize the qualitative detection of NT-proBNP, a biomarker of heart failure, in samples such as serum, plasma and urine by naked eyes, and can realize the quantitative detection of NT-proBNP based on the application of smart devices. It is a simple, rapid and sensitive detection method with broad application prospects.

[0009] This application provides the following technical solutions:

[0010] In a first aspect, the present application provides a lateral flow paper for detecting NT-proBNP, a biomarker of heart failure, the lateral flow paper comprising a bottom plate and a sample pad, a binding pad, a detection pad and a water-absorbing pad sequentially overlapped and pasted on the bottom plate along the sample chromatography direction;

[0011] The conjugate pad is coated with gold nanoparticle-coupled aptamer 1, and the aptamer 1 is an aptamer that can specifically bind to NT-proBNP;

[0012] The detection pad is coated with NT-proBNP-specific antibody and aptamer 2 at different positions to form a detection line and a quality control line respectively. The nucleotide sequence of the aptamer 2 is complementary to some bases of the aptamer 1, and the aptamer 2 is coupled with streptavidin.

[0013] Furthermore, the nucleotide sequence of the above aptamer 1 is shown in SEQ ID NO.1.

[0014] Furthermore, the nucleotide sequence of the above-mentioned aptamer 2 is shown in SEQ ID NO.2.

[0015] Specifically, the nucleotides and antibodies used in this application are shown in Table 1:

[0016] Table 1.

[0017]

[0018]

[0019] Furthermore, the preparation method of the gold nanoparticle-coupled aptamer 1 is as follows:

[0020] The thiolated aptamer 1 and the reducing agent are mixed and incubated for 0.5-1.5 h to obtain an activated aptamer 1 solution;

[0021] After adjusting the gold nanoparticle solution to alkalinity, the solution was mixed with the activated aptamer 1 solution, incubated for 1.5-2.5 hours, and then deoxyadenosine triphosphate disodium was added and incubated for 0.5-1.5 hours.

[0022] In the above technical scheme, the disulfide bond in the thiolated aptamer 1 is first reduced to a thiol group (-SH) by using a reducing agent. Since the thiol group has a strong affinity with the gold surface, the aptamer 1 can form a gold-sulfur bond with the surface of AuNPs through its thiol group, thereby stably attaching to the AuNPs. The purpose of adding disodium deoxyadenosine triphosphate for co-incubation is that the dATP residues on the AuNPs help reduce the nonspecific DNA binding to the particles through the nitrogenous base. The reduction of nonspecific binding by dATP helps to achieve the appropriate value of ssDNA in the subsequent detection process. In addition, the remaining space on the surface of Au-NP can be closed with dATP.

[0023] It is easier to form stable chemical bonds with the surface of gold nanoparticles.

[0024] Furthermore, the reducing agent is tris(2-carboxyethyl)phosphine, and the molar ratio of the thiolated aptamer 1 to the reducing agent is (45-55):1.

[0025] Furthermore, the molar ratio of the gold nanoparticles to the aptamer 1 and the deoxyadenosine disodium triphosphate is 1:(70-80):1000.

[0026] Furthermore, the conjugate pad is obtained by soaking the glass fiber in a conjugate buffer for 18-30 hours and then drying at 55-65° C. for 1-3 hours. The conjugate buffer is a PBS buffer containing 1-10% BSA, 1-10% sucrose and 0.1-0.5% Tween20.

[0027] Furthermore, the content of the gold nanoparticle-coupled aptamer 1 coated on the above-mentioned conjugate pad is: 0.002-0.003mmoL.

[0028] In a second aspect, the present application also provides a sensor for detecting the heart failure biomarker NT-proBNP, which comprises the above-mentioned lateral flow paper and an element for performing image acquisition and data analysis on the detection results of the lateral flow paper.

[0029] Furthermore, the detection range of the above sensor for NT-proBNP is 0.5-200 ng / mL.

[0030] In summary, this application has the following beneficial effects:

[0031] 1. The lateral flow paper provided by the present application is coated with a gold nanoparticle-coupled aptamer 1 as a binding pad, and NT-proBNP-specific antibodies and aptamers 2 are coated at different positions of the detection pad as detection lines and quality control lines, forming a lateral flow paper capable of specifically detecting NT-proBNP, a biomarker of heart failure. It has specific selectivity, low cost effectiveness and high efficiency, enabling it to be used for bedside (POC) analysis, especially when the most accurate results are needed in a short time (such as cardiovascular events).

[0032] 2. The sensor provided in the present application utilizes the above-mentioned lateral flow paper and an image acquisition and data analysis application on a smart device. By acquiring an image of the lateral flow paper after detection and analyzing the color intensity of the detection line on the image, the accurate concentration of NT-proBNP within the dynamic range in the biological sample can be obtained.

[0033] 3. Compared with in vivo prepared antibodies which are expensive, have limited immunogenicity and large batch differences, the nucleic acid aptamers used in this application have significant advantages such as easy generation, wide target range, easy modification, high binding affinity, high specificity, flexible structure, chemical synthesis modifiable, good biocompatibility and high stability. Although all reported NT-proBNP detection methods use antibodies, in the present invention, a new method is designed, which attempts to minimize the use of antibodies, using antibodies only as capture probes in the test line, combining aptamers with AuNPs, and also used as complementary DNA in the control pad, which has great advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a TEM image of the gold nanoparticles provided in Preparation Example 1 of this application;

[0035] Figure 2 UV-visible spectra of the three stages of the AuNPs modification process in Preparation Example 1 of this application;

[0036] Figure 3 A schematic diagram of the lateral flow paper for detecting the heart failure biomarker NT-proBNP provided in Example 1;

[0037] Figure 4 A schematic diagram of the coupling process of the lateral flow paper for detecting the heart failure biomarker NT-proBNP provided in Example 1;

[0038] Figure 5 A schematic diagram of the lateral flow paper provided in Example 1 in the image acquisition based on the smart device and the color intensity analysis test line;

[0039] Figure 6 This is the detection chromatography paper in which the NT-proBNP concentration increases from right to left during the detection limit determination process. DETAILED DESCRIPTION

[0040] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0041] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0042] Example of preparation of raw materials

[0043] Preparation Example 1

[0044] This preparation example provides a method for preparing a gold nanoparticle-coupled aptamer 1 (AuNPs-apt 1):

[0045] (1) Synthesis of gold nanoparticles: 100 mL of 0.01% HAuCl4 solution was boiled thoroughly, and then 2.2 mL of 1% (w / w) trisodium citrate solution was quickly added under vigorous stirring. The colloidal gold solution was boiled for another 6 min, and the color of the mixture turned into wine red within 3 min.

[0046] Figure 1 This is a TEM image of gold nanoparticles. As can be seen from the figure, the gold nanoparticles are regular circles with a particle size of 8-15nm.

[0047] (2) Preparation of AuNPs-apt 1:

[0048] 10mL of 10mmol / L thio-aptamer 1 (sequence: SEQ ID NO.1) and 2mL of 1mmol / L tris(2-carboxyethyl)phosphine (TCEP) were added to a polypropylene tube and incubated at room temperature for 1h to obtain activated aptamer 1. 1mL of AuNPs solution was adjusted to pH 8.0 with K2CO3 and concentrated to 0.1mL at 10000rpm. The activated aptamer 1 was transferred to the concentrated AuNPs solution and reacted at room temperature for 2h. Then, 9mL of 100mmol / L 2-deoxyadenosine 5-triphosphate disodium dihydrogen (dATP) was added and incubated for another 1h. The resulting mixture was added to a NaCl solution with a final concentration of 80mmol / L for aging.

[0049] After incubation at 4°C for 24 h, the unbound aptamer molecules were centrifuged at 10,000 rpm for 10 min at 4°C, and the conjugates were resuspended in 0.01 mol / L PBS (pH 7.4) containing 0.5% PEG-20000, 1% sucrose, 0.1% tween-20, 0.02% MgSO4, 0.05% (NH4)2SO4, and 1% BSA. Finally, AuNPs-apt1 was stored in a brown bottle at 4°C.

[0050] Figure 2 The UV-visible spectra of the three stages in the AuNPs modification process are shown in Figure 1. The blue line is AuNPs, the red line is AuNPs bound to aptamer 1 after aging in NaCl 80mM, and the green line is AuNPs bound to aptamer 1 and BSA. As can be seen from the figure, the maximum absorption of AuNPs is enhanced successively, and the maximum absorption wavelength also has a small red shift, which indicates that AuNPs-apt 1 is successfully prepared.

[0051] Preparation Example 2

[0052] This preparation example provides a preparation method of a gold nanoparticle-coupled aptamer 1 (AuNPs-apt 1), which is different from Preparation Example 1 in that (2) in the preparation of AuNPs-apt 1:

[0053] 10mL of 10mmol / L thio-aptamer 1 (sequence: SEQ ID NO.1) and 2mL of 1mmol / L tris(2-carboxyethyl)phosphine (TCEP) were added to a polypropylene tube and incubated at room temperature for 0.5h to obtain activated aptamer 1. 1mL of AuNPs solution was adjusted to pH 8.0 with K2CO3 and concentrated to 0.1mL at 10000rpm. The activated aptamer 1 was transferred to the concentrated AuNPs solution and reacted at room temperature for 2.5h. Then, 9mL of 100mmol / L 2-deoxyadenosine 5-triphosphate disodium dihydrogen (dATP) was added and incubated for another 1.5h. The resulting mixture was added to a NaCl solution with a final concentration of 80mmol / L for aging.

[0054] Preparation Example 3

[0055] This preparation example provides a preparation method of a gold nanoparticle-coupled aptamer 1 (AuNPs-apt 1), which is different from Preparation Example 1 in that (2) in the preparation of AuNPs-apt 1:

[0056] 10mL of 10mmol / L thio-aptamer 1 (sequence: SEQ ID NO.1) and 2mL of 1mmol / L tris(2-carboxyethyl)phosphine (TCEP) were added to a polypropylene tube and incubated at room temperature for 1.5h to obtain activated aptamer 1. 1mL of AuNPs solution was adjusted to pH 8.0 with K2CO3 and concentrated to 0.1mL at 10000rpm. The activated aptamer 1 was transferred to the concentrated AuNPs solution and reacted at room temperature for 1.5h. Then, 9mL of 100mmol / L 2-deoxyadenosine 5-triphosphate disodium dihydrogen (dATP) was added and incubated for another 0.5h. The resulting mixture was added to a NaCl solution with a final concentration of 80mmol / L for aging.

[0057] Preparation Example 4

[0058] This preparation example provides a method for preparing streptavidin-coupled aptamer 2 (SA-apt2), which comprises:

[0059] 5 μL of 1 mg / mL streptavidin solution (solvent: 0.01 M PBS, pH 7.4) and 5 μL of 10 μM aptamer 2 probe solution (sequence: SEQ ID NO. 2) were mixed and incubated at 37°C for 1 hour to allow for stable binding. After incubation at room temperature for 1 hour, 20 μL of PBS (0.01 M) was added.

[0060] Example 1

[0061] This embodiment provides a lateral flow paper and a sensor for detecting NT-proBNP, a biomarker of heart failure. Figure 3 The test strip consists of four key components: the sample pad and conjugate pad (glass fiber), the reaction pad (nitrocellulose membrane), and the absorption pad. These three elements are mounted on an adhesive backing card with a 2mm overlap to enable smooth fluid flow between the components.

[0062] The sample pad, nitrocellulose membrane, and absorbent pad were prepared as follows. They were then dried and stored in a low humidity chamber at room temperature.

[0063] (1) Preparation of sample pad:

[0064] The sample pad was soaked in a sample buffer, and then dried at 37°C and stored in a cool and ventilated place. The sample buffer was a PBS (0.01 M) solution containing 1% BSA, 0.2% Tween-20 and 2% sucrose.

[0065] (2) Preparation of conjugate pad:

[0066] The glass fiber was immersed in a conjugation buffer for 24 hours and then dried at 60° C. for 2 hours to obtain a PBS buffer containing 5% BSA, 5% sucrose and 0.3% Tween 20.

[0067] Then, AuNPs-apt 1 prepared in Preparation Example 1 was sprayed on the conjugate pad at a content of 0.0024 mmoL, thereby obtaining a conjugate pad coated with AuNPs-apt 1.

[0068] (3) Preparation of detection pad:

[0069] A detection line (T line) and a quality control line (C line) are arranged in parallel along the sample chromatography direction on the detection pad. The detection line is coated with NT-proBNP specific antibody (1 mg / mL, 1 μl / cm), and the quality control line is coated with SA-apt2 (0.0024 mmoL (80 mmol / L)) obtained in Preparation Example 4.

[0070] (4) Preparation of lateral flow chromatography paper:

[0071] The sample pad, conjugate pad, detection pad and absorbent pad were overlapped and pasted on the bottom plate in sequence, and cut into pieces with a width of 0.4 cm to obtain lateral flow paper.

[0072] (5) Detection of biological samples:

[0073] Take 50 μl of the sample to be tested and dissolve it in Tris buffer (Tris buffer 10 mM, 80 mM NaCl, pH 8, the concentration of the sample to be tested is 400 pg / ml-400 ng / ml), and add it dropwise to the sample pad. Based on capillary action, the sample liquid migrates from one end of the sample pad to the absorption pad. During the migration, it passes through the binding pad, and the NT-proBNP contained in the sample specifically binds to the AuNPs-apt 1 coated on the binding pad, and continues to migrate to the detection line, where it is captured by the NT-proBNP-specific antibody anchored on the detection line, and the detection line appears red; the free AuNPs-apt 1 that is not bound to NT-proBNP continues to migrate to the quality control line and is captured by the SA-apt2 anchored on the quality control line, and the quality control line appears red.

[0074] It usually takes 10 minutes from adding the sample to producing the color development result. In order to detect the accurate concentration, use a smartphone to take images and use the three primary colors colorimetric principle to perform data analysis to obtain the inspection results, such as Figure 4 , 5 shown.

[0075] Example 2-3

[0076] The difference between this group of examples and Example 1 lies in the preparation of the sample pad:

[0077] Example 2: The sample pad was soaked in a sample buffer, and then dried at 35° C. and stored in a cool and ventilated place. The sample buffer was a PBS (0.01 M) solution containing 0.2% BSA, 0.4% Tween-20 and 4% sucrose.

[0078] Example 3: The sample pad was soaked in a sample buffer, and then dried at 40° C. and stored in a cool and ventilated place. The sample buffer was a PBS (0.01 M) solution containing 2% BSA, 0.1% Tween-20 and 1% sucrose.

[0079] Embodiment 4-7

[0080] The difference between this group of examples and Example 1 lies in the preparation of the conjugate pad:

[0081] Example 4: The glass fiber was immersed in a conjugation buffer for 18 hours and then dried at 65° C. for 1 hour to obtain a conjugation buffer solution containing 10% BSA, 1% sucrose and 0.1% Tween 20 in a PBS buffer solution.

[0082] Example 5: The glass fiber was immersed in a conjugation buffer for 30 hours and then dried at 55° C. for 3 hours to obtain a conjugation buffer solution containing 1% BSA, 10% sucrose and 0.5% Tween 20 in a PBS buffer solution.

[0083] Example 6: The AuNPs-apt 1 prepared in Preparation Example 2 was sprayed on the conjugate pad at a content of 0.0024 mmoL (80 mmol / L), to obtain a conjugate pad coated with AuNPs-apt 1.

[0084] Example 7: The AuNPs-apt 1 prepared in Preparation Example 3 was sprayed on the conjugate pad at a content of 0.0024 mmoL (80 mmol / L) to obtain a conjugate pad coated with AuNPs-apt 1.

[0085] Detection limit determination:

[0086] The lateral flow paper provided in Example 1 was used to test a series of concentrations of NT-proBNP standard solutions:

[0087] The concentrations of NT-proBNP standard solutions are: 0, 0.5, 1, 6.25, 12.5, 25, 50, 100 and 200 ng / mL.

[0088] The results of the test are as follows Figure 6 As shown, Figure 6 The NT-proBNP concentration of the detection chromatographic paper increases from right to left. It can be seen that for the NT-proBNP standard solution with a concentration of 0, the detection line has no color, while for the NT-proBNP standard solution with a concentration of 0.5 ng / mL, the detection line appears red visible to the naked eye. As the concentration increases, the color becomes more obvious. It can be seen that the lateral chromatography paper provided by the present application has high sensitivity and a detection range of 0.5-200 ng / mL.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A lateral flow paper for detecting NT-proBNP, a biomarker of heart failure, characterized in that: The lateral flow paper is composed of a bottom plate and a sample pad, a binding pad, a detection pad and a water-absorbing pad which are overlapped and pasted on the bottom plate in sequence along the sample chromatography direction; The conjugate pad is coated with gold nanoparticle-coupled aptamer 1, and the aptamer 1 is an aptamer that can specifically bind to NT-proBNP; The detection pad is coated with NT-proBNP-specific antibody and aptamer 2 at different positions to form a detection line and a quality control line respectively. The nucleotide sequence of the aptamer 2 is complementary to some bases of the aptamer 1, and the aptamer 2 is coupled with streptavidin.

2. The lateral flow paper for detecting the heart failure biomarker NT-proBNP according to claim 1, characterized in that: The nucleotide sequence of the aptamer 1 is shown in SEQ ID NO.

1.

3. The lateral flow paper for detecting the heart failure biomarker NT-proBNP according to claim 2, characterized in that: The nucleotide sequence of the aptamer 2 is shown in SEQ ID NO.

2.

4. The lateral flow paper for detecting the heart failure biomarker NT-proBNP according to claim 1, characterized in that: The preparation method of the gold nanoparticle-coupled aptamer 1 is as follows: The thiolated aptamer 1 and the reducing agent are mixed and incubated for 0.5-1.5 h to obtain an activated aptamer 1 solution; After adjusting the gold nanoparticle solution to alkalinity, the solution was mixed with the activated aptamer 1 solution, incubated for 1.5-2.5 hours, and then deoxyadenosine disodium triphosphate was added and incubated for 0.5-1.5 hours.

5. The lateral flow paper for detecting the heart failure biomarker NT-proBNP according to claim 4, characterized in that: The reducing agent is tris(2-carboxyethyl)phosphine, and the molar ratio of the thiolated aptamer 1 to the reducing agent is (45-55):

1.

6. The lateral flow paper for detecting the heart failure biomarker NT-proBNP according to claim 4, characterized in that: The molar ratio of the gold nanoparticles to the aptamer 1 and the deoxyadenosine disodium triphosphate is 1:(70-80):1000.

7. The lateral flow paper for detecting the heart failure biomarker NT-proBNP according to claim 1, characterized in that: The conjugate pad is obtained by soaking the glass fiber in a conjugation buffer for 18-30 hours and then drying at 55-65° C. for 1-3 hours. The conjugation buffer is a PBS buffer containing 1-10% BSA, 1-10% sucrose and 0.1-0.5% Tween 20.

8. The lateral flow paper for detecting the heart failure biomarker NT-proBNP according to claim 1, characterized in that: The content of the gold nanoparticle-coupled aptamer 1 coated on the conjugate pad is 0.002-0.003 mmoL.

9. A sensor for detecting NT-proBNP, a biomarker of heart failure, characterized in that: The lateral flow paper comprises the lateral flow paper according to any one of claims 1 to 8, and a component for performing image acquisition and data analysis on the detection results of the lateral flow paper.

10. The sensor for detecting the heart failure biomarker NT-proBNP according to claim 9, characterized in that: The sensor has a detection range of 0.5-200 ng / mL for NT-proBNP.

Images