阅读说明：本技术 一种检测深海环境基因的电化学传感系统及检测方法 (Electrochemical sensing system and method for detecting deep sea environment genes ) 是由 雷建平 朱达 王思娜 李俊 付昊旻 于 2020-11-26 设计创作，主要内容包括：本发明涉及一种检测深海环境基因的电化学传感系统及检测方法,其目的在于以二茂铁标记的探针作为直接式信号,利用碱基互补配对的原则,通过目标基因诱导探针构型发生变化,改变二茂铁与电极表面的距离产生电流响应,借助三段式封装设计使其实现深海核酸的原位高通量检测。三段式封装设计包括工作站主板舱、线路连接舱和检测舱；含目标基因的细胞裂解液流经检测舱,与传感器探针发生杂交反应,实现目标基因检测；使电化学传感器件能够搭载着陆器进入深海正常、自主运行,实现深海极端环境下的核酸原位检测；二茂铁标记的探针作为直接式信号,无需外加信号来源；无试剂参与,简单易操作。(The invention relates to an electrochemical sensing system and a detection method for detecting deep sea environmental genes, aiming at using a ferrocene-labeled probe as a direct signal, utilizing the principle of base complementary pairing, inducing the probe configuration to change by a target gene, changing the distance between ferrocene and the surface of an electrode to generate current response, and realizing the in-situ high-flux detection of deep sea nucleic acid by means of a three-section packaging design. The three-section type packaging design comprises a workstation main board cabin, a line connection cabin and a detection cabin; enabling cell lysate containing target genes to flow through the detection cabin and perform hybridization reaction with the sensor probe to realize target gene detection; the electrochemical sensing device can be carried with a lander to enter deep sea to normally and automatically operate, and the in-situ detection of nucleic acid under the extreme environment of deep sea is realized; the ferrocene-labeled probe is used as a direct signal without an additional signal source; no reagent is involved, and the method is simple and easy to operate.)

1. An electrochemical sensing system for detecting deep sea environment genes is characterized in that the specific packaging design is as follows: the structure of the system comprises a workstation main board cabin, a line connection cabin and a detection cabin; enabling cell lysate containing target genes to flow through the detection cabin and perform hybridization reaction with the sensor probe to realize target gene detection;

the main board cabin of the workstation is packaged by adopting a dry cabin, an L-shaped platform is arranged in a cabin cover, and the micro electrochemical workstation is fixedly installed; pore channels are reserved at two ends of the cabin body, the micro electrochemical workstation is respectively connected with the sensing interface and the upper computer through watertight inserts, and the cabin body and the cabin cover are sealed through watertight gaskets; the middle circuit connection cabin is packaged by a wet cabin; the detection cabin comprises an electrode sensing interface part and a liquid flow part to be detected; the parts are sealed by sealing gaskets, and the electrodes are sealed by sealing rings.

2. The electrochemical sensing system for detecting deep sea environmental genes as claimed in claim 1, wherein said workstation main board compartment body is cylindrical and disperses the pressure under water.

3. The electrochemical sensing system for detecting deep sea environmental genes as claimed in claim 1, wherein said circuit connection cabin body is provided with oil holes for adding hydraulic oil, connecting compensation oil bag and exhausting gas in the cabin.

4. The electrochemical sensing system for detecting deep sea environmental genes as claimed in claim 1, wherein the electrode sensing interface portion comprises a working electrode, a reference electrode and an auxiliary electrode.

5. The electrochemical sensing system for detecting deep sea environmental genes as claimed in claim 4, wherein the working electrode is a probe-modified gold electrode.

6. The electrochemical sensing system for detecting deep sea environmental genes as claimed in claim 1, wherein the detecting chamber electrode copper rod end is provided with a rear cover fixing part to ensure that the working electrode is located on a plane higher than the reference and auxiliary electrodes, thereby preventing the electrodes from moving backwards during polishing.

7. The electrochemical sensing system for detecting the deep sea environment genes as claimed in claim 1, wherein the system carries a lander to enter deep sea to normally and autonomously operate, so that nucleic acid in-situ detection under the deep sea extreme environment is realized.

8. The electrochemical sensing system for detecting deep sea environmental genes as claimed in claim 1, wherein the detection method comprises constructing a direct signal reagent-free DNA electrochemical sensor on the surface of an electrode, and inducing the probe configuration to change by using a target gene to generate current response, thereby realizing in-situ detection of nucleic acid under the deep sea extreme environment; the method specifically comprises the following steps:

1) centrifuging ferrocene-labeled probe dry powder at a high speed, and adding a buffer solution to obtain a probe solution with a certain concentration;

2) oscillating and activating the probe solution obtained in the step 1) and tris (2-carbonyl ethyl) phosphate for later use, wherein the oscillation time is 1 hour;

3) polishing the surface of the gold electrode until the mirror surface is smooth;

4) dripping the activated probe solution prepared in the step 2) on the surface of the electrode polished in the step 3) to fix the probe on the surface of the electrode;

5) repeatedly washing the surface of the electrode for 2-3 times, and dropwise adding a proper amount of 6-mercaptohexane-1-alcohol to seal unreacted electrode sites to obtain an electrode for modifying the probe; installing a modified probe electrode in a direct signal electrochemical sensor detection cabin;

6) dropwise adding a target gene reaction solution with a certain concentration on the surface of an electrode of a modified probe, carrying out hybridization reaction on a target gene and the probe to form a three-chain structure, and changing the distance between a ferrocene molecule on the probe and the surface of the electrode so as to change a current signal;

7) in a three-electrode system, obtaining a current signal response curve, and reading a peak current after processing; and making a working curve of the target gene standard solution, and calculating the target gene concentration at the sampling position of the deep sea extreme environment through the working curve.

Technical Field

The invention discloses an electrochemical sensing system and a detection method for detecting deep sea environment genes, and belongs to the technical field of electrochemical sensing.

Background

The functional gene information of deep-sea microorganisms or larvae is obtained, expression information and protein activity characteristics of the functional genes in an in-situ environment are revealed through omics analysis, and the comprehensive and three-dimensional understanding of deep-sea life from cell genes can be realized. At present, the detection of deep sea functional genes is mainly carried out in a land laboratory based on sampling of a sampling bottle, and the change of the surrounding environment seriously influences the analysis result. Therefore, it is urgent to design a sensor device and method for in situ detection of functional gene fragments of deep-sea microorganisms or larvae.

The direct signal reagent-free electrochemical sensor has the advantages of low price, easy obtaining, one-step detection and the like, and meanwhile, the chip sensor can be used for field detection. Such sensors are primarily based on the interaction of nucleic acids with small molecules, nucleic acids, nanomaterials, etc. to design signal switches. Due to the convenience of the direct signal reagent-free electrochemical sensor, the application range of the direct signal reagent-free electrochemical sensor is very wide. In daily life, the reagent can detect ochratoxin A in food; in bioassays, it can detect cocaine in whole blood. Therefore, the characteristics of simplicity and rapidness are fully utilized, and the software control and instrument packaging design are combined, so that the method is applied to the nucleic acid in-situ detection in the deep sea environment, and a technical support is provided for revealing the functional analysis on the microbiocoenosis level under the deep sea in-situ condition.

Disclosure of Invention

The invention provides an electrochemical sensing system and a detection method for detecting deep sea environmental genes, aiming at using a ferrocene-labeled probe as a direct signal, utilizing the principle of base complementary pairing, inducing the probe configuration to change by a target gene, changing the distance between ferrocene and the surface of an electrode to generate current response, and realizing the in-situ high-flux detection of deep sea nucleic acid by means of a three-section packaging design.

The technical solution of the invention is as follows:

taking a ferrocene-labeled probe as a direct signal probe, carrying out oscillation reaction on the direct signal probe and tris (2-carbonyl ethyl) phosphate for 1 hour, taking out a proper amount of mixed solution, and dropwise adding the mixed solution on the surface of an electrode to assemble a single-layer probe molecule; then washing and dripping a proper amount of 6-mercaptohexan-1-ol to seal unreacted sites. In the presence of a target gene, the target gene and a probe sequence form a triple-stranded structure according to a base complementary pairing principle, so that the distance between a ferrocene molecule and the surface of an electrode is changed, the electron transfer rate is changed, and finally the current is represented on the level of the magnitude of the current. The type of the target gene can be qualitatively judged by the current, and the target gene quantification purpose can be achieved by combining a standard working curve.

Wherein, the tris (2-carbonyl ethyl) phosphate is used as a direct signal probe for further activation; the whole detection process is carried out in a three-electrode system.

A direct signal reagent-free DNA electrochemical sensor is constructed on the surface of an electrode, and a target gene is utilized to induce the probe configuration to change so as to generate current response, so that the in-situ detection of nucleic acid under the deep sea extreme environment is realized, and the method specifically comprises the following steps:

1) centrifuging ferrocene-labeled probe dry powder at a high speed, and adding a buffer solution to obtain a probe solution with a certain concentration;

2) oscillating and activating the probe solution obtained in the step 1) and tris (2-carbonyl ethyl) phosphate for later use, wherein the oscillation time is 1 hour;

3) polishing the surface of the gold electrode until the mirror surface is smooth;

4) dripping the activated probe solution prepared in the step 2) on the surface of the electrode polished in the step 3) to fix the probe on the surface of the electrode;

5) repeatedly washing the surface of the electrode for 2-3 times, and dropwise adding a proper amount of 6-mercaptohexane-1-alcohol to seal unreacted electrode sites to obtain an electrode for modifying the probe; installing a modified probe electrode in a direct signal electrochemical sensor detection cabin;

6) dropwise adding a target gene reaction solution with a certain concentration on the surface of an electrode of a modified probe, carrying out hybridization reaction on a target gene and the probe to form a three-chain structure, and changing the distance between a ferrocene molecule on the probe and the surface of the electrode so as to change a current signal;

7) in a three-electrode system, obtaining a current signal response curve, and reading a peak current after processing; and making a working curve of the target gene standard solution, and calculating the target gene concentration at the sampling position of the deep sea extreme environment through the working curve.

The direct signal electrochemical sensor is specifically packaged and designed as follows when applied to a deep sea environment:

s1: the main board cabin of the workstation is packaged by a dry cabin, and the cabin body of the dry cabin is cylindrical; an L-shaped platform is arranged in the hatch cover, and a miniature electrochemical workstation is fixedly arranged; pore channels are reserved at two ends of the cabin body, the micro electrochemical workstation is respectively connected with the sensing interface and the upper computer through watertight inserts, and the cabin body and the cabin cover are sealed through watertight gaskets;

s2: the middle line connection cabin is packaged by a wet cabin, and the cabin body is provided with an oil hole for adding hydraulic oil, connecting a compensation oil bag and exhausting gas in the cabin;

s3: the detection cabin comprises an electrode sensing interface part and a liquid flow part to be detected; the parts are sealed by sealing gaskets, and the electrodes are sealed by sealing rings.

The detection method principle of the device is as follows:

the electrochemical sensing device constructs a soft interface based on the specific recognition of the target gene induced configuration change. The probe for marking the ferrocene molecules is fixed on the surface of the electrode through covalent bond action, and at the moment, the ferrocene molecules are far away from the surface of the electrode, so that the electron transfer rate is slow and the current value is small. When the target gene exists, a triple-chain structure is formed through the double recognition effect of Watson-Crick and Hoogsteen base pairing, the ferrocene molecule is drawn to the surface of the electrode, the electron transfer rate is high, the current value is increased, and the current signal shows regular change along with the gradual increase of the concentration of the target gene. On the other hand, the electrochemical workstation and the sensing interface are integrated and sealed in the cabin body, so that the aim of in-situ detection of the target gene in the deep sea environment is fulfilled.

The invention has the beneficial effects that:

1) the ferrocene-labeled probe is used as a direct signal without an additional signal source; no reagent is involved, and the method is simple and easy to operate;

2) the system adopts a three-section type packaging method, so that an electrochemical sensing device can be carried with a lander to enter deep sea to normally and automatically operate, and the in-situ detection of nucleic acid under the extreme environment of deep sea is realized;

3) the workstation mainboard is cylindrical dry chamber, can disperse the pressure under water, and the pressure of liquid makes the unable vibration of mainboard crystal oscillator in the wet cabin, loses signal transmission's function and leads to the unable work of mainboard.

Drawings

FIG. 1 is a schematic diagram of in-situ detection of deep sea environment genes based on a direct electrochemical sensor device.

FIG. 2 is a schematic diagram of three-stage packaging of an electrochemical sensing system for detecting deep sea environmental genes.

In the figure, 1 is a working electrode, 2 is a reference electrode, and 3 is an auxiliary electrode.

Detailed Description

The technical solution of the present invention is further explained with reference to the accompanying drawings.

Referring to the attached figure 2, the packaging design of the electrochemical sensing system for detecting deep sea environment genes is divided into three sections (figure 2), the leftmost side is a main board of the miniature electrochemical workstation and is packaged by a dry cabin, a left cabin cover is a watertight cabin penetrating piece to lead out a power supply, a working lead and the like, and the left cabin cover is connected with an upper computer to execute the functions of command, signal or data transmission, file storage and the like. The working station main board with the wet cabin packaged in the middle is connected with the biological sensing interface through a circuit, and oil holes are processed on the surface of the cabin body. The rightmost side is a liquid cabin through which cell lysate containing target genes flows and is also a reaction cabin of the reagent-free electrochemical biosensor, and the target genes and the sensor probes are subjected to hybridization reaction to realize target gene detection.

After the device is built, a rear cover fixing piece is added at the end of the electrode copper bar. On one hand, the working electrode can be fixed, so that the electrode is prevented from moving backwards during polishing, and polishing is facilitated; on the other hand, the plane of the working electrode is higher than the plane of the reference and auxiliary electrodes, the tiger fish acid solution is favorable for treating the working electrode, and the electrode can be well reused without being detached.

Referring to FIG. 1, the working principle of the present invention is that a probe as a direct signal and tris (2-carbonylethyl) phosphate are reacted for 1 hour by shaking, a proper amount of the probe is taken out and dripped on the surface of an electrode to assemble a monolayer probe molecule, and then a proper amount of 6-mercaptohexane-1-ol is washed and dripped to seal unreacted sites. The probe for marking the ferrocene molecules is fixed on the surface of the electrode through covalent bond action, and at the moment, the ferrocene molecules are far away from the surface of the electrode, so that the electron transfer rate is slow and the current value is small. When the target gene exists, a triple-chain structure is formed through the double recognition effect of Watson-Crick and Hoogsteen base pairing, the ferrocene molecule is drawn to the surface of the electrode, the electron transfer rate is high, the current value is increased, and the current signal shows regular change along with the gradual increase of the concentration of the target gene. The type of the target gene can be qualitatively judged by the current, and the target gene quantification purpose can be achieved by combining a standard working curve.

The specific detection steps are as follows:

1) and ensuring that the plane of the working electrode is higher than the planes of the reference electrode and the auxiliary electrode, dripping wet aluminum oxide powder on the surface of the chamois, coating the chamois on the electrode plane, slightly applying force, turning and polishing, washing with clear water after polishing, and performing ultrasonic treatment for 2-3 minutes. Placing the treated electrode at 1M H₂SO₄Activating for standby.

2) mu.L of 10. mu.M ferrocene-labeled probe and 50. mu.L of 10 mM Tris (2-carbonylethyl) phosphate and 40. mu.L of 25 mM Tris-HCl 200 mM Na were taken⁺After mixing evenly, shaking for reaction for 1 hour for standby.

3) And dropwise adding 5-8 mu L of the activated probe on the surface of the dried and packaged working electrode-gold electrode, and reacting for 2 hours at normal temperature. Then washing with clear water, drying by blowing, dripping 5 mu L of 1 mM 6-mercaptohexane-1-alcohol for reaction for about 1 hour, and then washing away the redundant 6-mercaptohexane-1-alcohol for later use.

4) Preparing standard solutions with different concentrations, testing the response of the sensing device to the standard sample, and drawing a corresponding standard working curve. The constructed gene sensing device is carried at the downstream of the seawater filtering device and is detected in cell lysate containing target genes to obtain the target gene.

5) And dropwise adding 5-8 mu L of the activated probe on the surface of the dried and packaged working electrode-gold electrode, and reacting for 2 hours at normal temperature. Then washing with clear water, drying by blowing, dripping 5 mu L of 1 mM 6-mercaptohexane-1-alcohol for reaction for about 1 hour, and then washing away the redundant 6-mercaptohexane-1-alcohol for later use.

6) Preparing standard solutions with different concentrations, testing the response of the sensing device to the standard sample, and drawing a corresponding standard working curve. The constructed gene sensing device is arranged at the downstream of the seawater filtering device, and is used for detecting in cell lysate containing target genes to obtain a current signal response curve, and the concentration of the target genes in the sample is obtained from the working curve.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.