Blood glucose detection method based on nucleic acid aptamer

Abstract

The invention discloses a blood glucose detection method based on a nucleic acid aptamer. The blood glucose detection method comprises the following steps: S1, constructing a compound; s2, realizing fluorescence signal change; s3, quantitatively analyzing the glucose concentration. The invention relates to the technical field of biological detection, in particular to a blood glucose detection method based on an aptamer, which has the following advantages: 1, high stability and low cost; 2, high specificity; 3, multifunctionality and expansibility are realized; and 4, determining a wide detection range and a linear relation.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a blood glucose detection method based on nucleic acid aptamers. Background Art

[0002] Blood glucose testing is crucial for the diagnosis, treatment, and monitoring of diabetes and for the study of metabolic diseases. Traditional methods for blood glucose testing, such as enzymatic methods (glucose oxidase), rely on enzyme-catalyzed reactions, which can lead to issues such as enzyme inactivation, poor stability, and high costs. While nanomaterial-based sensor technology has advanced, existing sensors often rely on antibodies or enzymes as their recognition units, which can be challenging, including insufficient specificity, complex preparation, and poor environmental adaptability.

[0003] Aptamers are single-stranded nucleic acids (DNA or RNA) that can specifically bind to target molecules, resulting from screening. They offer advantages such as high stability, low cost, and ease of chemical modification. However, how to utilize aptamers to construct highly sensitive blood glucose sensors, particularly through structural design to achieve efficient signal conversion, remains a pressing technical challenge in this field. Summary of the Invention

[0004] In view of this, the present invention aims to provide a blood glucose detection method based on nucleic acid aptamers, which utilizes the structural transformation of nucleic acid aptamers after binding to glucose, combined with the fluorescence quenching characteristics of nanomaterials, to achieve high-specificity and high-sensitivity detection of glucose, and solve the problems of poor stability of recognition units and high detection costs in the existing technology.

[0005] The technical solution of the embodiment of the present invention is achieved as follows:

[0006] A blood glucose detection method based on nucleic acid aptamers comprises the following steps:

[0007] S1. Complex construction: incubating the fluorescently labeled glucose aptamer with the nanomaterial to form an aptamer-nanomaterial complex, wherein the aptamer is adsorbed on the surface of the nanomaterial through base stacking force;

[0008] S2. Achieving fluorescence signal change: In the buffer system, the complex emits no fluorescence signal due to the fluorescence quenching effect of the nanomaterial; after adding glucose, the nucleic acid aptamer specifically binds to glucose and forms a secondary or tertiary structure, detaches from the surface of the nanomaterial, and the fluorescence signal is restored;

[0009] S3. Quantitative analysis of glucose concentration: Quantitative analysis of glucose concentration was performed by detecting changes in fluorescence signal intensity.

[0010] Preferably, the nanomaterial includes graphene oxide, carbon nanotubes, carbon nanoparticles, fullerenes, gold nanoparticles, gold nanorods, silver nanoparticles or silver nanorods, which are used to adsorb nucleic acid aptamers and quench fluorescence signals.

[0011] Preferably, the sequence of the glucose aptamer is: 5'-FAM-CTC TCG ACG ACC GTG TGT GTTGCT CTG TAA CAG TGT CCA TTG TCG TC-3', its core structure comprises a hairpin-macroloop-hairpin-smallloop structure, and the terminal loop sequence can be increased or decreased to optimize the binding performance.

[0012] Preferably, the buffer system is: 500 mM NaCl, 10 mM KCl, 10 mM MgCl2, 50 mM HEPES, pH 7.4-7.5.

[0013] Preferably, the detection signal in S3 includes a fluorescence signal, a fluorescence polarization signal, a Raman signal, a phosphorescence signal, an electrochemical signal, an electrochemiluminescence signal or a visual detection signal.

[0014] Preferably, the nucleic acid aptamer is a single-chain structure before binding to glucose, and is converted into a complex binary structure after binding.

[0015] The tertiary structure, the structural conversion is used to trigger signal output, including fluorescence signal conversion based on hairpin probes or visualization signal conversion based on nanoparticle dispersion / aggregation.

[0016] Preferably, the hairpin probe is designed as follows: a complementary sequence is introduced at one end of the nucleic acid aptamer to form a hairpin structure, and the ends are labeled with a fluorescent molecule and a quenching group respectively. After binding to glucose, the structure unfolds, the fluorescent molecule and the quenching group are separated, and the fluorescence signal is restored.

[0017] Preferably, the visualization detection based on nanoparticle dispersion / aggregation is designed as follows: when the single-stranded nucleic acid aptamer is adsorbed on the surface of gold nanoparticles, gold nanorods, silver nanoparticles or silver nanorods, the nanoparticles are in a dispersed state (the solution is red); after binding to glucose, the nucleic acid aptamer is detached, the nanoparticles aggregate, and the color of the solution changes to blue / black.

[0018] Preferably, the core structure of the glucose nucleic acid aptamer is hairpin-macroloop-hairpin-small loop, comprising at least two hairpin structures and one macroloop structure, allowing the binding activity to be adjusted by increasing or decreasing the terminal loop sequence while maintaining the base pairing pattern of the core recognition region.

[0019] Preferably, the detection range of the method is 0-50 mM, and it has a linear response relationship within the concentration range of 2-50 mM, and is suitable for quantitative detection of human blood glucose samples.

[0020] The embodiment of the present invention adopts the above technical solution, which has the following advantages:

[0021] 1. High stability and low cost: DNA aptamers replace traditional enzyme recognition units, are acid and alkali resistant, high temperature resistant, have a long shelf life, and significantly reduce preparation costs.

[0022] 2. High specificity: The specific binding of nucleic acid aptamers to glucose is based on three-dimensional structural matching, which avoids possible cross-reactions of antibodies or enzymes and has high detection accuracy.

[0023] 3. Versatility and scalability: There is a wide range of nanomaterials to choose from (carbon-based materials, precious metal nanomaterials, etc.), and the output signal forms are diverse (fluorescence, electrochemistry, visualization, etc.), which can be adapted to different detection scenarios (laboratory instrument detection or portable visualization detection).

[0024] 4. Wide detection range and linear relationship: Experiments have shown that it has a good linear relationship in the concentration range of 2-50mM, which can cover the common concentration range of clinical blood glucose testing (0-50mM) and is suitable for human blood glucose testing.

[0025] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or technical descriptions. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is the full flow chart of the core detection of the present invention;

[0028] Figure 2 This is a signal output branch flow chart of the present invention;

[0029] Figure 3 This is the full flow chart of clinical sample testing of the present invention;

[0030] Figure 4 Establish a process flow chart for the standard curve of the present invention;

[0031] Figure 5 The figure is a flow chart comparing the applications of different nanomaterials of the present invention. DETAILED DESCRIPTION

[0032] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and description are to be considered as illustrative in nature and not restrictive.

[0033] It should be noted that the terms "first," "second," "symmetrical," "array," etc. are used only to distinguish descriptions from positional descriptions and should not be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, limitations on features such as "first" and "symmetrical" may explicitly or implicitly include one or more of these features; similarly, when the number of certain features is not limited in the form of words such as "two" or "three," it should be noted that these features also explicitly or implicitly include one or more of the number of features.

[0034] In the present invention, unless otherwise expressly specified or limited, terms such as "installation," "connection," and "fixation" should be understood broadly; for example, they may refer to fixed connection, detachable connection, or integral molding; they may refer to mechanical connection, direct connection, welding, or indirect connection through an intermediate medium; they may refer to internal communication between two components or interaction between two components. Those skilled in the art will be able to understand the specific meanings of the above terms in the present invention based on the specification and drawings in conjunction with specific circumstances.

[0035] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0036] like Figure 1-5 The present invention provides a method for detecting blood glucose based on nucleic acid aptamers, comprising the following steps:

[0037] S1. Complex construction: Fluorescently labeled glucose aptamers are incubated with nanomaterials to form aptamer-nanomaterial complexes. The aptamers are adsorbed on the surface of the nanomaterials through base stacking forces.

[0038] S2. Achieving fluorescence signal change: In the buffer system, the complex emits no fluorescence signal due to the fluorescence quenching effect of the nanomaterial; after adding glucose, the nucleic acid aptamer specifically binds to glucose and forms a secondary or tertiary structure, detaches from the surface of the nanomaterial, and the fluorescence signal is restored;

[0039] S3. Quantitative analysis of glucose concentration: Quantitative analysis of glucose concentration was performed by detecting changes in fluorescence signal intensity.

[0040] like Figure 1-5As shown, the nanomaterials include graphene oxide, carbon nanotubes, carbon nanoparticles, fullerenes, gold nanoparticles, gold nanorods, silver nanoparticles or silver nanorods, which are used to adsorb nucleic acid aptamers and quench fluorescence signals.

[0041] like Figure 1-5 As shown, the sequence of the glucose aptamer is: 5'-FAM-CTC TCG ACG ACC GTG TGT GTTGCT CTG TAA CAG TGT CCA TTG TCG TC-3', its core structure comprises a hairpin-macroloop-hairpin-miniloop structure, and the terminal loop sequence is allowed to be increased or decreased to optimize the binding performance. The core structure of the glucose aptamer is hairpin-macroloop-hairpin-miniloop, comprising at least two hairpin structures and one macroloop structure, allowing the binding activity to be adjusted by increasing or decreasing the terminal loop sequence, while maintaining the base pairing pattern of the core recognition region.

[0042] like Figure 1-5 As shown, the buffer system is: 500 mM NaCl, 10 mM KCl, 10 mM MgCl2, 50 mM HEPES, pH 7.4-7.5.

[0043] like Figure 1-5 As shown, the detection signal in S3 includes a fluorescence signal, a fluorescence polarization signal, a Raman signal, a phosphorescence signal, an electrochemical signal, an electrochemiluminescence signal or a visual detection signal.

[0044] like Figure 1-5 As shown, the nucleic acid aptamer is a single-stranded structure before binding to glucose, and is converted into a complex secondary / tertiary structure after binding. The structural conversion is used to trigger signal output, including fluorescence signal conversion based on hairpin probes or visualization signal conversion based on nanoparticle dispersion / aggregation. The design based on hairpin probes is as follows: a complementary sequence is introduced at one end of the nucleic acid aptamer to form a hairpin structure, and the ends are labeled with fluorescent molecules and quenching groups respectively. After binding to glucose, the structure unfolds, the fluorescent molecules and the quenching groups are separated, and the fluorescence signal is restored. The visualization detection design based on nanoparticle dispersion / aggregation is as follows: when the single-stranded nucleic acid aptamer is adsorbed on the surface of gold nanoparticles, gold nanorods, silver nanoparticles or silver nanorods, the nanoparticles are dispersed (the solution is red); after binding to glucose, the nucleic acid aptamer is detached, the nanoparticles aggregate, and the color of the solution changes to blue / black.

[0045] like Figure 1-5 As shown, the detection range of the method is 0-50 mM, and it has a linear response relationship in the concentration range of 2-50 mM, which is suitable for the quantitative detection of human blood glucose samples.

[0046] In this embodiment, the present invention is specifically designed to work as follows:

[0047] First, the complex is constructed: a fluorescently labeled glucose aptamer solution (sequence such as 5'-FAM-CTCTCG ACG ACC GTG TGT GTT GCT CTG TAA CAG TGT CCA TTG TCG TC-3') is mixed with a nanomaterial (such as graphene oxide) in a certain proportion and incubated at a suitable temperature (such as 25°C) for a period of time (such as 30 minutes). The aptamer is adsorbed to the graphene oxide surface through base stacking forces, forming an aptamer-nanomaterial complex. The solution is then centrifuged (such as at 10,000 rpm for 5 minutes) to remove unadsorbed aptamers and ensure the purity of the complex.

[0048] The detection system was then prepared and the signal was triggered: the complex was placed in a buffer solution (500mM NaCl, 10mM KCl, 10mM MgCl2, 50mM HEPES, pH 7.4). At this point, the system emitted no fluorescent signal due to fluorescence quenching by graphene oxide. Adding a certain concentration of glucose solution caused specific binding of glucose to the aptamer, causing the aptamer to fold into a secondary structure and detach from the graphene oxide surface, restoring the fluorescent signal.

[0049] Finally, the concentration is tested: the fluorescence signal intensity of the system is detected using a fluorescence detector. The concentration of the glucose solution to be tested is determined by comparative analysis using a pre-established standard curve of fluorescence signal intensity versus glucose concentration (detection range 0-50mM, linear response within 2-50mM).

[0050] The following are several other specific embodiments of the present invention:

[0051] Example 1: Clinical blood glucose detection

[0052] Sample preparation stage: Collect blood samples from patients in the hospital, and obtain serum or plasma samples through centrifugation and other methods to ensure that the samples are free of impurities and contamination and meet the testing requirements.

[0053] During the test phase, fluorescently labeled glucose aptamers are incubated with gold nanoparticles to form a complex, which is then placed in a specific buffer system. A certain amount of treated serum sample is added to the system. Glucose binds to the aptamers, triggering a change in fluorescence signal. The intensity of the fluorescence signal is rapidly measured using specialized fluorescence detection equipment.

[0054] Results Application Phase: The detected fluorescence signal intensity is converted into a blood glucose concentration value based on a pre-established standard curve. Doctors use this blood glucose value, combined with the patient's other clinical indicators, to diagnose diabetes, adjust treatment plans, or monitor the patient's condition.

[0055] Example 2: Rapid blood glucose monitoring at home

[0056] Preparation stage: Use a pre-prepared detection kit (containing the formed nucleic acid aptamer-nanomaterial complex and buffer) and ensure that the storage conditions of the kit meet the requirements (such as low temperature and avoid light).

[0057] Detection phase: The user collects a small amount of fingertip blood and, following the kit instructions, adds the blood sample to the kit's reaction system. The glucose in the system binds to the aptamer, triggering a change in the fluorescence signal.

[0058] Results acquisition: A small, accompanying testing device (such as a portable fluorescence detector) reads the fluorescence signal and automatically converts it into a blood glucose concentration value, which is displayed on the screen. This allows users to understand their blood glucose levels in real time, allowing them to adjust their diet, exercise, or seek medical attention promptly.

[0059] Example 3: Large-scale blood glucose screening

[0060] Sample processing stage: In the community or physical examination center, a large number of blood samples are collected from the subjects and pre-processed uniformly to ensure the consistency and standardization of the samples.

[0061] Batch testing: Using automated testing equipment, pre-treated samples are sequentially added to the testing system (containing the aptamer-nanomaterial complex and buffer). The equipment automatically detects changes in the fluorescence signal intensity of each sample.

[0062] Data Analysis: Using the accompanying data analysis software, the fluorescence signal intensity is converted into blood glucose concentration data, allowing rapid screening of individuals with abnormal blood glucose levels. For those identified as high-risk, detailed medical examinations are arranged to achieve early detection and intervention for diabetes.

[0063] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various modifications and substitutions within the technical scope disclosed in the present invention, and such modifications and substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A blood glucose detection method based on nucleic acid aptamers, characterized in that: The following steps are involved: S1. Complex construction: incubating the fluorescently labeled glucose aptamer with the nanomaterial to form an aptamer-nanomaterial complex, wherein the aptamer is adsorbed on the surface of the nanomaterial through base stacking force; S2. Achieving fluorescence signal change: In the buffer system, the complex emits no fluorescence signal due to the fluorescence quenching effect of the nanomaterial; after adding glucose, the nucleic acid aptamer specifically binds to glucose and forms secondary and tertiary structures, detaches from the surface of the nanomaterial, and the fluorescence signal is restored; S3. Quantitative analysis of glucose concentration: Quantitative analysis of glucose concentration was performed by detecting changes in fluorescence signal intensity.

2. The method for detecting blood glucose based on nucleic acid aptamers according to claim 1, wherein: The nanomaterials include graphene oxide, carbon nanotubes, carbon nanoparticles, fullerenes, gold nanoparticles, gold nanorods, silver nanoparticles or silver nanorods, which are used to adsorb nucleic acid aptamers and quench fluorescence signals.

3. The method for detecting blood glucose based on nucleic acid aptamers according to claim 1, wherein: The sequence of the glucose aptamer is: 5'-FAM-CTC TCG ACG ACC GTG TGT GTT GCT CTG TAA CAG TGT CCATTG TCG TC-3', and its core structure includes a hairpin, a macroloop, a hairpin and a microloop structure, and the terminal loop sequence can be increased or decreased to optimize the binding performance.

4. The method for detecting blood glucose based on nucleic acid aptamers according to claim 1, wherein: The buffer system is: 500mM NaCl, 10mM KCl, 10mM MgCl2, 50mM HEPES, pH 7.4-7.

5.

5. The method for detecting blood glucose based on nucleic acid aptamers according to claim 1, wherein: The detection signal in S3 includes a fluorescence signal, a fluorescence polarization signal, a Raman signal, a phosphorescence signal, an electrochemical signal, an electrochemiluminescence signal or a visual detection signal.

6. The method for detecting blood glucose based on nucleic acid aptamers according to claim 1, wherein: The nucleic acid aptamer is a single-chain structure before binding to glucose, and is converted into complex secondary and tertiary structures after binding. The structural conversion is used to trigger signal output, including fluorescent signal conversion based on hairpin probes or visual signal conversion based on nanoparticle dispersion and aggregation.

7. The method for detecting blood glucose based on nucleic acid aptamers according to claim 6, wherein: The hairpin probe is designed as follows: a complementary sequence is introduced at one end of the nucleic acid aptamer to form a hairpin structure, and the ends are labeled with a fluorescent molecule and a quenching group respectively. After binding to glucose, the structure unfolds, the fluorescent molecule and the quenching group separate, and the fluorescence signal is restored.

8. The method for detecting blood glucose based on nucleic acid aptamers according to claim 6, wherein: The visualization detection design based on nanoparticle dispersion and aggregation is as follows: when the single-stranded nucleic acid aptamer is adsorbed on the surface of gold nanoparticles, gold nanorods, silver nanoparticles or silver nanorods, the nanoparticles are in a dispersed state; after binding to glucose, the nucleic acid aptamer is detached, the nanoparticles aggregate, and the color of the solution changes to blue and black.

9. The method for detecting blood glucose based on nucleic acid aptamers according to claim 3, wherein: The core structure of the glucose nucleic acid aptamer is a hairpin, a macroloop, a hairpin and a microloop, comprising at least two hairpin structures and one macroloop structure, allowing the binding activity to be adjusted by increasing or decreasing the terminal loop sequence while maintaining the base pairing pattern of the core recognition region.

10. A blood glucose detection method based on nucleic acid aptamers according to any one of claims 1 to 9, characterized in that: The detection range of the method is 0-50 mM, and it has a linear response relationship within the concentration range of 2-50 mM, and is suitable for quantitative detection of human blood glucose samples.

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