Electrochemical biosensor for detecting mycoplasma pneumoniae

Abstract

The invention provides an electrochemical biosensor for detecting mycoplasma pneumoniae, the core of the electrochemical biosensor comprises a specific capture probe, and the nucleotide sequence of the specific capture probe is a complementary sequence of a 23S-5S rDNA spacer sequence (ITS) of the mycoplasma pneumoniae. In the preparation link, the porous membrane is used as a high-efficiency electron transmission channel, the process is remarkably simplified, and the reaction efficiency is improved. By virtue of a capture probe taking 23S-5S rDNA ITS as a target, the sensor is endowed with excellent specificity and high sensitivity, so that the sensor has excellent performance in complex biological sample detection, can accurately and efficiently recognize a target object and effectively avoids interference of other substances. The electrochemical biosensor is easy and convenient to operate and high in response speed, has wide application prospects in the fields of clinical diagnosis and rapid disease screening, can provide key technical support for timely diagnosis of mycoplasma pneumoniae infection, greatly meets the urgent requirements for rapid and accurate detection in clinical and practical application, and has good application prospects. The development process of a biological detection technology in the mycoplasma pneumoniae detection direction is powerfully promoted.

Description

Technical Field

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

[0002] The size of Mycoplasma pneumoniae (MP) is between that of bacteria and viruses. It is the smallest cell with the shortest gene fragment discovered so far. As one of the common pathogens of respiratory tract infection, it is widely spread around the world, especially 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 infection to severe pneumonia and other disease manifestations. It is contagious to a certain extent, which poses a challenge to public health and air defense. Traditional methods for detecting Mycoplasma pneumoniae, such as culture, serological detection and nucleic acid amplification technology, have problems such as long detection cycle, complex operation and limited sensitivity. Electrochemical biosensors, with their advantages of rapidity, sensitivity and simplicity, have shown great potential in the field of biomolecule detection. Therefore, the establishment of an efficient and accurate electrochemical biosensor for MP detection is of great significance to improve the cure rate, reduce the overuse of antibiotics and alleviate the suffering of patients.

[0003] As an 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 methods, 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 a recognition target. The 23S-5S rDNA ITS of Mycoplasma pneumoniae is conservative and specific in this pathogen. Its complementary sequence is used as a capture probe to accurately identify Mycoplasma pneumoniae and avoid nonspecific binding with other pathogen nucleic acids. It provides an ideal choice for the development of highly sensitive and specific electrochemical biosensors, which is expected to fill the gaps in traditional detection methods, meet the urgent needs of rapid and accurate detection of Mycoplasma pneumoniae in clinical and on-site settings, and promote the further development of respiratory infection disease diagnosis technology. It has 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 easy to operate and has a fast response speed. It 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 of biological detection technology in the direction of Mycoplasma pneumoniae detection.

[0005] The present invention provides an electrochemical biosensor for detecting Mycoplasma pneumoniae, and a respiratory disease diagnosis platform is constructed with 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 double electrode system, and a capture probe with a complementary sequence of 23S-5S rDNA ITS is connected in the nanopores of the porous membrane. The electrochemical sensor involved in the present invention uses a porous membrane as an electron transmission channel, has a simple preparation process and high reaction efficiency, and 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 is an electrochemical biosensor for detecting Mycoplasma pneumoniae, characterized by comprising:

[0007] (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';

[0008] (b) a substrate material having a nanostructure, used for fixing the capture probe;

[0009] (c) A signal detection unit, used to detect the signal change generated after the capture probe binds to Mycoplasma pneumoniae.

[0010] The base material is a porous membrane with nanopores, the pore size of the nanopores is 10nm-10μm, and the channel length is 5-25μm.

[0011] The capture probes are fixed to the substrate material in a manner including, but not limited to, chemical covalent bonds, physical adsorption, and self-assembled monolayer technology.

[0012] The signal detection unit detects Mycoplasma pneumoniae by detecting at least one of the following parameters: impedance change, current response, potential change, and capacitance change.

[0013] The technical principle of the present invention is:

[0014] When the test sample containing Mycoplasma pneumoniae comes into contact with the porous membrane, its 23S-5S rDNA ITS will hybridize with the specific capture probe in the nanopore of the porous membrane according to the principle of base complementary pairing to form a stable double-stranded nucleic acid structure. 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, 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 a known concentration in advance, the detected electrochemical signal can be converted into the actual concentration of Mycoplasma pneumoniae in the sample, thereby achieving quantitative detection of Mycoplasma pneumoniae.

[0015] During the entire 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 electrochemical signals. The biosensor of the present invention includes a porous membrane and a recognition unit composed of capture probes on its nanopores. These components work together to ensure the accuracy and reliability of the sensor in detecting Mycoplasma pneumoniae, providing strong support for the diagnosis of respiratory diseases, and is expected to play an important role in clinical diagnosis, epidemic prevention and control, and other fields, and promote technological progress and development in related industries.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] 1. The capture probe can specifically identify the target sequence of Mycoplasma pneumoniae and accurately identify the target. Combined with the high-sensitivity detection principle of electrochemical biosensors, low-concentration Mycoplasma pneumoniae can be detected.

[0018] 2. The entire detection process does not require complicated sample pretreatment and long-term culture process. It can be completed within 45 minutes from sample addition to result output, which can meet the needs of clinical diagnosis.

[0019] 3. The complementary DNA of Mycoplasma pneumoniae 23S-5S rDNA ITS is used as the biological recognition element, which is highly specific to Mycoplasma pneumoniae and can effectively avoid interference from other pathogens or impurities, ensuring the accuracy of the test results.

[0020] 4. The sensor has a relatively simple structure and is easy to operate. It does not require professional operators and complex instruments and equipment, and is easy to promote and apply in primary medical institutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary 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 accompanying drawings:

[0022] Figure 1Schematic diagram of the detection principle of an electrochemical biosensor for the detection of Mycoplasma pneumoniae

[0023] Figure 2 Optimization of (A) probe concentration and (B) Mycoplasma pneumoniae incubation time during electrochemical biosensor construction

[0024] Figure 3 IV curves of cTnI responses at different concentrations

[0025] Figure 4 Standard curve of the current change rate curve in response to different concentrations of cTnI

[0026] Figure 5 Histograms of assay stability, temporal stability, and specificity DETAILED DESCRIPTION

[0027] Example 1

[0028] A method for preparing an electrochemical biosensor for detecting Mycoplasma pneumoniae, wherein the method is as follows Figure 1 As shown, the following steps are included:

[0029] (1) The nuclear pore membrane is placed in a mixed solution prepared with MES buffer for activation. The mixed solution contains 10 mg / mL N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 1 mg / mL N-hydroxysulfosuccinimide (NHS). In this process, the carboxyl groups in the nanopores are activated into amide functional groups.

[0030] (2) The activated nuclear pore membrane was then immersed in a solution with a concentration of 8 μM ( Figure 2 A) was incubated in the amino capture probe solution for 45 min ( Figure 2 B), so that the capture probe can effectively bind to the activated nuclear pore membrane. After incubation, the functionalized nuclear pore membrane is washed with MES buffer solution to remove unbound capture probes and other impurities. Then, bovine serum albumin is used to block the active site to prevent nonspecific adsorption.

[0031] Example 2

[0032] An application of an electrochemical biosensor for Mycoplasma pneumoniae detection as described above comprises the following steps:

[0033] The functionalized nuclear pore membrane in Example 1 was used as the middle diaphragm of the H-type electrolytic cell. Subsequently, the electrolyte solution was injected into the two chambers of the electrolytic cell respectively, and then the electrodes were placed in the corresponding chambers to construct a complete electrochemical system. After that, under the application of a certain voltage, the change of the current was monitored by IV detection means, and the electrochemical current was recorded. At this time, the current was recorded as I0 ( Figure 3 );

[0034] The electrochemical biosensor was placed in a test solution of Mycoplasma pneumoniae with different concentrations for incubation, and then rinsed with phosphate buffer several times to remove Mycoplasma pneumoniae that was not specifically bound to the sensor. After the rinse was completed, the sensor was tested and the current value at this time was recorded and marked as I. The relative change rate of the current before and after the addition of Mycoplasma pneumoniae was calculated by the electrochemical workstation to achieve quantitative detection of the target, and a standard curve with Mycoplasma pneumoniae concentration and current relative change rate (I0-I) / I0 was drawn. Figure 4 ).

[0035] Under the same conditions, the current value of the same electrochemical biosensor was measured 7 times in succession ( Figure 5 A), with a relative standard deviation (RSD) of about 1.03%; after storage at 4°C, 95.13% of the response current value was retained after 13 days of storage compared with the first day ( Figure 5 B).

[0036] Example 3

[0037] In order to explore the specificity of this detection method for Mycoplasma pneumoniae, the standard solutions of Chlamydia pneumoniae, respiratory syncytial virus, metapneumovirus and rhinovirus were used as controls, and the experiment was carried out according to the detection method of Example 2. The results are as follows: Figure 5 C. For the interfering substances Chlamydia pneumoniae, respiratory syncytial virus, metapneumovirus and rhinovirus, the relative change rates of the current in the detection system were maintained at (6.47±1.2)%, (9.12±1.7)%, (4.40±1.0)% and (3.13±1.8)%, respectively. When the detection target was Mycoplasma pneumoniae, the current change rate of the entire system was significantly increased to (25.45±2.16)%. The experimental results intuitively show that this detection method has a high specificity for Mycoplasma pneumoniae and can effectively distinguish Mycoplasma pneumoniae from other common respiratory pathogens, providing a strong guarantee for the accurate detection of Mycoplasma pneumoniae.

[0038] The above description is only the best embodiment of the present invention and is not intended to limit the spirit and principle of the present invention. Any modifications, equivalent substitutions, improvements, etc. made should 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 having a nanostructure, used for fixing the capture probe; (c) A signal detection unit, used to detect the signal change generated after the capture probe binds to Mycoplasma pneumoniae.

2. An electrochemical biosensor for detecting Mycoplasma pneumoniae according to claim 1, characterized in that: The base material is a porous membrane with nanopores, the pore size of the nanopores is 10nm-10μm, and the channel length is 5-25μm.

3. The electrochemical biosensor for detecting Mycoplasma pneumoniae according to claim 1, characterized in that: The capture probes are fixed to the substrate material in a manner including, but not limited to, chemical covalent bonds, physical adsorption, and self-assembled monolayer technology.

4. The electrochemical biosensor for detecting Mycoplasma pneumoniae according to claim 1, characterized in that: The signal detection unit detects Mycoplasma pneumoniae by detecting at least one of the following parameters: impedance change, current response, potential change, and capacitance change.

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