An electrochemical biosensor for the detection of Mycoplasma pneumoniae

Abstract

The present invention provides an electrochemical biosensor for the detection of Mycoplasma pneumoniae. The core includes a specific capture probe, and its nucleotide sequence is the complementary sequence of the 23S-5S rDNA internal transcribed spacer (ITS) of Mycoplasma pneumoniae. In the preparation process, a porous membrane is used as an efficient electron transfer channel, significantly simplifying the process and improving the reaction efficiency. With the capture probe targeting 23S-5S rDNA ITS, the sensor is endowed with excellent specificity and high sensitivity, enabling it to perform outstandingly in the detection of complex biological samples, accurately and efficiently identifying the target substance and effectively avoiding interference from other substances. The electrochemical biosensor of the present invention is easy to operate and has a fast response speed, with broad application prospects in the fields of clinical diagnosis and rapid disease screening. It can provide key technical support for the timely diagnosis of Mycoplasma pneumoniae infection, greatly meeting the urgent need for rapid and accurate detection in clinical and practical applications, and strongly promoting the development process of biological detection technology in the direction of Mycoplasma pneumoniae detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical biosensors, and particularly relates to an electrochemical biosensor for detecting Mycoplasma pneumoniae. Background Art

[0002] Mycoplasma pneumoniae (MP) has a size between bacteria and viruses. It is the smallest cell organism with the shortest gene fragment discovered so far. As one of the common pathogenic bacteria causing respiratory tract infections, it spreads widely globally, posing a serious threat to the health of children, adolescents, and people with low immunity. The symptoms caused by its infection range from mild upper respiratory tract infections to severe pneumonia and other disease manifestations, and it is contagious, bringing challenges to public health prevention and control. Traditional detection methods for Mycoplasma pneumoniae, such as culture methods, serological tests, and nucleic acid amplification techniques, have problems such as long detection cycles, complex operations, and limited sensitivity. Electrochemical biosensors have shown great potential in the field of biomolecule detection due to their advantages of rapidity, sensitivity, and simplicity. Therefore, establishing an efficient and accurate electrochemical biosensor for MP detection is of great significance for improving the cure rate, reducing the overuse of antibiotics, and alleviating the pain of patients.

[0003] As a new emerging biological detection technology, electrochemical biosensors have shown great potential in the field of biomolecule detection due to their advantages of rapidity, sensitivity, simple operation, and relatively low cost. By combining biorecognition elements with electrochemical detection means, rapid and specific detection of target molecules can be achieved. In the detection of Mycoplasma pneumoniae, it is of great significance to use its unique nucleic acid sequence as the recognition target. The 23S-5S rDNA ITS of Mycoplasma pneumoniae is conserved and specific in this pathogen. Using its complementary sequence as a capture probe can accurately identify Mycoplasma pneumoniae, avoiding non-specific binding with nucleic acids of other pathogens, providing an ideal choice for developing highly sensitive and highly specific electrochemical biosensors, promising to fill the deficiencies of traditional detection methods, meet the urgent needs of clinical and on-site rapid and accurate detection of Mycoplasma pneumoniae, promote the further development of diagnostic technologies for respiratory tract infectious diseases, and have important application value and practical significance in disease prevention and control, clinical treatment decision-making, and public health monitoring. Summary of the Invention

[0004] The electrochemical biosensor of the present invention is extremely simple to operate and has a fast response speed, and has broad application prospects in clinical diagnosis and rapid disease screening. It can provide key technical support for the timely diagnosis and prevention and control of Mycoplasma pneumoniae infection, greatly meet the urgent needs for rapid and accurate detection in clinical and practical applications, and strongly promote the development process of biological detection technology in the direction of Mycoplasma pneumoniae detection.

[0005] The present invention provides an electrochemical biosensor for detecting Mycoplasma pneumoniae, and thereby constructs a respiratory disease diagnosis platform using the complementary sequence of Mycoplasma pneumoniae 23S-5S rDNA ITS as a specific recognition element. The electrochemical biosensor includes a porous membrane and an electrochemical two-electrode system, and a capture probe with the complementary sequence of 23S-5S rDNA ITS is connected inside the nanopores of the porous membrane. The electrochemical sensor involved in the present invention uses the porous membrane as an electron transfer channel, and the preparation process is simple and the reaction efficiency is high. The electrochemical biosensor constructed based on this has high sensitivity and high selectivity, and can be applied to the detection of Mycoplasma pneumoniae in complex biological samples. The biosensor includes a substrate and a recognition unit composed of capture probes adsorbed on the substrate.

[0006] The present invention relates to an electrochemical biosensor for detecting Mycoplasma pneumoniae, which is characterized by comprising: (a) a specific capture probe, the nucleotide sequence of which is the complementary sequence of the Mycoplasma pneumoniae 23S-5S rDNA spacer sequence: 5'-GGAAGCCTTTGGTAGGAAATACGCAGG-3';

[0007] (b) a substrate material with nanostructures for immobilizing the capture probe;

[0008] (c) a signal detection unit for detecting the signal change generated after the capture probe binds to Mycoplasma pneumoniae.

[0009] The substrate material is a porous membrane with nanopores, and the pore diameter of the nanopores is 10 nm to 10 μm, and the channel length is 5 to 25 μm.

[0010] The ways of immobilizing the capture probe on the substrate material include but are not limited to chemical covalent bonds, physical adsorption, and self-assembled monolayer technology.

[0011] The signal detection unit realizes the detection of Mycoplasma pneumoniae by detecting at least one of the following parameters: impedance change, current response, potential change, and capacitance change.

[0012] The technical principle of the present invention is:

[0013] When a test sample containing Mycoplasma pneumoniae contacts the porous membrane, its 23S-5S rDNA ITS hybridizes with the specific capture probe in the nanopores of the porous membrane according to the base complementary pairing principle, forming a stable double-stranded nucleic acid structure. Since the hybridization of Mycoplasma pneumoniae with the capture probe changes the pore size of the porous membrane and thus changes the flux of the system, a measurable change in the electrochemical signal is generated, which has a certain correlation with the concentration of Mycoplasma pneumoniae in the sample. By establishing a standard curve with a Mycoplasma pneumoniae standard of known concentration in advance, the detected electrochemical signal can be converted into the actual concentration of Mycoplasma pneumoniae in the sample, thus realizing the quantitative detection of Mycoplasma pneumoniae.

[0014] During the whole detection process, the porous membrane not only serves as a fixed carrier for the capture probe, but its unique nanopore structure also plays a key role in the generation and transmission of the electrochemical signal. The biosensor of the present invention covers an identification unit composed of a porous membrane and a capture probe on its nanopores. These components cooperate with each other to jointly ensure the accuracy and reliability of the sensor for the detection of Mycoplasma pneumoniae, providing strong support for the diagnosis of respiratory diseases, and is expected to play an important role in the fields of clinical diagnosis, epidemic prevention and control, etc., promoting the technological progress and development of related industries.

[0015] Compared with the prior art, the beneficial effects of the present invention are at least as follows:

[0016] 1. By specifically recognizing the target sequence of Mycoplasma pneumoniae with the capture probe, the target can be accurately recognized. Combining with the high-sensitivity detection principle of the electrochemical biosensor, the detection of low-concentration Mycoplasma pneumoniae can be realized.

[0017] 2. The whole detection process does not require complex sample pretreatment and long-time cultivation process. The result can be output within 45 minutes from the sample addition, which can meet the needs of clinical diagnosis.

[0018] 3. Using the complementary DNA of Mycoplasma pneumoniae 23S-5S rDNA ITS as a biological recognition element, it has high specificity for Mycoplasma pneumoniae, can effectively avoid the interference of other pathogens or impurities, and ensure the accuracy of the detection result.

[0019] 4. The structure of the sensor is relatively simple, easy to operate, does not require professional operators and complex instrument equipment, and is easy to be popularized and applied in primary medical institutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0021] Figure 1Detection schematic diagram of an electrochemical biosensor for detecting Mycoplasma pneumoniae

[0022] Figure 2 Optimization of (A) probe concentration and (B) Mycoplasma pneumoniae incubation time during the construction of the electrochemical biosensor

[0023] Figure 3 I-V curves of responses to different concentrations of Mycoplasma pneumoniae

[0024] Figure 4 Standard curve of the current change rate curves of responses to different concentrations of Mycoplasma pneumoniae

[0025] Figure 5 Bar charts of detection stability, time stability and specificity Detailed implementation manners

[0026] Example 1

[0027] A preparation method of an electrochemical biosensor for detecting Mycoplasma pneumoniae, and its implementation method is as Figure 1 shown, including the following steps:

[0028] (1) Place the nuclear pore membrane in a mixed solution prepared with MES buffer for activation. This mixed solution contains N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) with a concentration of 10 mg / mL and N-hydroxysulfosuccinimide (NHS) with a concentration of 1 mg / mL. During this process, the carboxyl groups in the nanopores are activated into amide functional groups.

[0029] (2) Subsequently, immerse the activated nuclear pore membrane in an amino-functionalized capture probe solution with a concentration of 8 μM ( Figure 2 A) and incubate for 45 min ( Figure 2 B) to effectively bind the capture probe to the activated nuclear pore membrane. After incubation, wash the functionalized nuclear pore membrane with MES buffer solution to remove unbound capture probes and other impurities. Subsequently, use bovine serum albumin to block the active sites to prevent non-specific adsorption.

[0030] Example 2

[0031] An application of an electrochemical biosensor for detecting Mycoplasma pneumoniae as described above includes the following steps:

[0032] 1) Use the functionalized nuclear pore membrane in Example 1 as the middle diaphragm of the H-type electrolytic cell. Subsequently, inject the electrolyte solution into the two chambers of the electrolytic cell respectively, and then place the electrodes in the corresponding chambers to construct a complete electrochemical system. After that, under a certain applied voltage, monitor the change of current by means of I-V detection means and record the electrochemical current. At this time, the current is recorded as I0( Figure 3 );

[0033] 2) Incubate the electrochemical biosensor in the Mycoplasma pneumoniae test solution with different concentrations, and then rinse it with phosphate buffer solution multiple times to remove the Mycoplasma pneumoniae that has not specifically bound to the sensor. After rinsing, test the sensor and record the current value at this time, marked as I. By calculating the relative change rate of the current before and after adding Mycoplasma pneumoniae detected by the electrochemical workstation, the quantitative detection of the target substance is realized, and a standard curve with the concentration of Mycoplasma pneumoniae and the relative change rate of current (I0-I) / I0 on the horizontal and vertical coordinates is plotted( Figure 4 ).

[0034] 3) Under the same conditions, measure the current value of the same electrochemical biosensor 7 times continuously( Figure 5 A), and its relative standard deviation (RSD) is about 1.03%; store it at 4°C. Compared with the first day, after storing for 13 days, 95.13% of the response current value is retained( Figure 5 B).

[0035] Example 3

[0036] To explore the specificity of this detection method for Mycoplasma pneumoniae, use the standard solution of Chlamydia pneumoniae, Respiratory syncytial virus, Metapneumovirus and Rhinovirus as controls, and conduct experiments according to the detection method in Example 2. The obtained results are as Figure 5 shown in C. For the interfering substances Chlamydia pneumoniae, Respiratory syncytial virus, Metapneumovirus and Rhinovirus, the relative change rates of the current in the detection system are maintained at (6.47±1.2)%, (9.12±1.7)%, (4.40±1.0)% and (3.13±1.8)% respectively. When the detection target is Mycoplasma pneumoniae, the current change rate of the whole system is significantly increased to (25.45±2.16)%. The experimental results intuitively show that this detection method has high specificity for Mycoplasma pneumoniae, can effectively distinguish Mycoplasma pneumoniae from other common respiratory pathogens, and provides a strong guarantee for the accurate detection of Mycoplasma pneumoniae.

[0037] The above is only the best embodiment of the present invention, and it is not intended to limit the spirit and principle of the present invention. Any modifications, equivalent replacements, improvements, etc. made shall be included in the protection scope of the present invention.

Claims

1. An electrochemical biosensor for detecting Mycoplasma pneumoniae, characterized in that: Include: (a) a specific capture probe, the nucleotide sequence of which is the complementary sequence of the 23S-5S rDNA spacer sequence of Mycoplasma pneumoniae: 5'-GGAAGCCTTTGGTAGGAAATACGCAGG-3'; (b) a substrate material with a nanostructure, used to fix the capture probe; the substrate material is a porous membrane with nanopores, the pore size of the nanopores is 10 nm to 10 μm, and the channel length is 5 to 25 μm; (c) A signal detection unit, which is used to detect the signal change generated after the capture probe binds to Mycoplasma pneumoniae; since the hybridization between Mycoplasma pneumoniae and the capture probe changes the pore size of the porous membrane and thus changes the flux size of the system, the electrochemical signal produces a measurable change.

2. An electrochemical biosensor for detecting Mycoplasma pneumoniae according to claim 1, characterized in that: The capture probe is fixed to the base material by chemical covalent bonding.

3. The electrochemical biosensor for detecting Mycoplasma pneumoniae according to claim 1, characterized in that: The signal detection unit detects Mycoplasma pneumoniae by detecting the following parameters: current response.

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