Integrated exosome capture and analysis method based on responsive DNA nanostructures

Abstract

Disclosed is an integrated exosome capture and analysis method based on responsive DNA nanostructures. The method comprises the following steps: step one, constructing a detection system; step two, fabricating a PDMS chip; step three, arranging the detection system; step four, performing sample pretreatment; step five, performing exosome capture; and step six, performing exosome analysis. A multi-microchannel microfluidic chip is used as an exosome capture and analysis site, and a detection system is arranged on the microfluidic chip, wherein a capture module of the detection system uses responsive DNA nanostructures to capture exosomes, and uses a magnetic field to accelerate the capture process; and an analysis module of the detection system interprets marker expression levels of the exosomes by means of combined electrical and fluorescence signals. Compared with existing detection methods, the present invention not only reduces detection costs, sample consumption and operational complexity, but also improves the detection speed and the accuracy of a detection result, thereby facilitating automated detection of large-scale samples.

Description

An integrated method for exosome capture and analysis based on responsive DNA nanostructures Technical Field

[0001] This invention relates to the field of molecular biology, specifically to an integrated method for exosome capture and analysis based on responsive DNA nanostructures. Background Technology

[0002] Exosomes are a class of extracellular vesicles containing specific proteins, nucleic acids, and lipids. Exosomes produced by cancer cells serve as a form of intercellular communication, promoting cell growth and increasing tumor invasiveness and metastasis. In recent years, liquid biopsy has gained widespread attention as a non-invasive screening method, and exosomes, as potential biomarkers, show strong clinical application prospects in cancer diagnosis and treatment. In existing technologies, tumor exosome detection methods based on responsive DNA nanostructures mainly involve constructing a DNA hydrogel through double rolling circle amplification to capture exosomes in cell supernatant. The captured exosomes bind through bispecific recognition, triggering a CHA reaction to form a large amount of dsDNA. This dsDNA can be used as a target for CRISPR / Cas12a, activating its unique trans-cleavage activity to cleave fluorescent probes. Specific and sensitive detection of the target is achieved by detecting the fluorescence signal, thereby analyzing exosomes. This method has the following drawbacks: First, the detection cost is high. The reagents and equipment required for exosome capture analysis are expensive, and a large sample volume is needed, making it unsuitable for situations with limited sample sizes. Second, the detection speed is slow. The capture and analysis process is time-consuming, failing to meet the needs of rapid detection. Furthermore, it is difficult to achieve high-throughput detection because only one sample can be processed at a time, making it unsuitable for large-scale sample detection. Additionally, the use of a single fluorescence signal for data analysis is not conducive to improving the accuracy and reliability of the analytical results. Third, the operation is technically challenging. The entire detection process involves many steps and requires professional technicians to operate, demanding a high level of technical expertise from the operators. Summary of the Invention

[0003] The purpose of this invention is to provide an integrated method for exosome capture and analysis based on responsive DNA nanostructures, in order to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: an integrated method for exosome capture and analysis based on responsive DNA nanostructures, comprising the following steps: Step 1, constructing a detection system; Step 2, manufacturing a PDMS chip; Step 3, deploying the detection system; Step 4, sample pretreatment; Step 5, exosome capture; Step 6, exosome analysis;

[0005] In step one above, the constructed detection system includes a capture module and an analysis module. The capture module includes a capture unit, a capture enhancement unit, a control unit, and a signal detection unit. The analysis module includes an electrochemical analysis unit, an image analysis unit, a data interpretation unit, and a result output unit. The signal detection unit includes an electrical signal detection subunit and a fluorescence signal detection subunit.

[0006] In step two above, a multi-microchannel positive mold is fabricated using photolithography, and then a PDMS chip semi-finished product is fabricated using the multi-microchannel positive mold.

[0007] In step three above, a capture module and an analysis module are arranged on the PDMS chip semi-finished product made in step two, and the PDMS chip semi-finished product is bonded to a glass plate to obtain the PDMS chip finished product.

[0008] In step four above, the sample containing exosomes is pretreated to remove impurities and cell debris from the sample.

[0009] In step five above, the preprocessed sample from step four is injected into the PDMS chip product from step three. The exosomes are captured by the capture module, and the exosome information is collected by the signal detection unit and sent to the analysis module.

[0010] In step six above, the analysis module analyzes the exosome information and outputs the analysis results.

[0011] Preferably, in step two, the process of fabricating a multi-microchannel template using photolithography involves: spin-coating photoresist onto a silicon wafer, pre-baking and then placing it in a photolithography machine, covering it with a mask and exposing it, and post-baking followed by development with a developing solution to obtain a multi-microchannel positive mold.

[0012] Preferably, in step two, the process of fabricating a PDMS chip semi-finished product using a multi-microchannel template is as follows: PDMS and curing agent are mixed at a mass ratio of 10:1, then poured onto a multi-microchannel positive mold. After removing air bubbles by vacuuming, the mixture is placed in an oven and dried at 95°C for 30 minutes. The cured PDMS is then peeled off from the multi-microchannel positive mold to obtain the PDMS chip semi-finished product.

[0013] Preferably, in step three, the arrangement of the capture module and the analysis module specifically involves: depositing an electrochemical detection electrode on the multi-microchannel surface of the PDMS chip semi-finished product as an electrochemical signal detection subunit; fixing a responsive DNA nanostructure on the surface of the electrochemical detection electrode to form a capture unit; installing an electromagnet on the chip to form a capture enhancement unit; establishing an electrical connection between the capture enhancement unit and the control unit; establishing an electrical connection between the electrochemical signal detection subunit and the electrochemical analysis unit; establishing an electrical connection between the fluorescence signal detection subunit and the image analysis unit; establishing a data connection between both the image analysis unit and the electrochemical analysis unit and the data interpretation unit; and establishing a data connection between the data interpretation unit and the result output unit, thereby completing the arrangement of the capture module and the analysis module.

[0014] Preferably, the responsive DNA nanostructure incorporates a fluorescent probe.

[0015] Preferably, the electrochemical detection electrode includes a working electrode, a counter electrode, and a reference electrode.

[0016] Preferably, the immobilization of the responsive DNA nanostructure on the surface of the electrochemical detection electrode specifically involves: modifying the responsive DNA nanostructure with thiol groups, and then dropping the thiol-modified DNA nanostructure solution onto the surface of the working electrode, thereby immobilizing the responsive DNA nanostructure on the working electrode.

[0017] Preferably, in step five, the capture of exosomes by the capture module and the collection of exosome information by the signal detection unit to the analysis module specifically involves: using the capture unit to capture exosomes in the sample; simultaneously, the control unit controls the magnetic field strength and direction of the capture enhancement unit to accelerate the capture process; and the signal detection unit uses the electrical signal detection subunit to collect electrical signals to the electrochemical analysis unit and uses the fluorescence signal detection subunit to collect fluorescence images to the image analysis unit.

[0018] Preferably, the capture unit captures exosomes in the sample by: the responsive DNA nanostructure specifically binding to the marker on the surface of the exosome, exciting the fluorescent probe to generate a fluorescent signal, and at the same time, the electrochemical detection electrode collects the electrical signal.

[0019] Preferably, in step six, the analysis module analyzes the exosome information and outputs the analysis results as follows: the image analysis unit preprocesses the detected fluorescence image using an image analysis algorithm, then analyzes and extracts fluorescence intensity and fluorescence distribution information, and sends the results to the data interpretation unit; the electrochemical analysis unit filters the detected electrical signal, then analyzes and extracts current information, and sends the results to the data interpretation unit; the data interpretation unit integrates current information, fluorescence intensity, and fluorescence distribution information to interpret the marker expression level of exosomes; and the result output unit outputs the interpretation results in the form of charts.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention uses a multi-microchannel microfluidic chip as the site for exosome capture and analysis. The microfluidic chip is equipped with a detection system. The capture module of the detection system uses responsive DNA nanostructures to capture exosomes and uses a magnetic field to accelerate the capture process. The analysis module of the detection system interprets the expression level of exosome markers by combining electrical and fluorescence signals. Compared with existing detection methods, this invention not only reduces detection costs, sample consumption, and operational difficulty, but also improves detection speed and accuracy of detection results, which is conducive to realizing automated detection of large-scale samples. Attached Figure Description

[0021] Figure 1 is a flowchart of the method of the present invention;

[0022] Figure 2 is a block diagram of the detection system of the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Please refer to Figures 1 and 2. One embodiment of the present invention provides an integrated method for exosome capture and analysis based on responsive DNA nanostructures, comprising the following steps: Step 1, constructing a detection system; Step 2, manufacturing a PDMS chip; Step 3, deploying the detection system; Step 4, sample pretreatment; Step 5, exosome capture; Step 6, exosome analysis.

[0025] In step one above, the constructed detection system includes a capture module and an analysis module. The capture module includes a capture unit, a capture enhancement unit, a control unit, and a signal detection unit. The analysis module includes an electrochemical analysis unit, an image analysis unit, a data interpretation unit, and a result output unit. The signal detection unit includes an electrical signal detection subunit and a fluorescence signal detection subunit.

[0026] In step two above, a multi-microchannel positive mold is fabricated using photolithography. Specifically, photoresist is spin-coated onto a silicon wafer, pre-baked, and then placed in a photolithography machine. A mask is covered and exposed, followed by post-baking and development with a developer to obtain the multi-microchannel positive mold. Then, the multi-microchannel positive mold is used to fabricate a PDMS chip semi-finished product. Specifically, PDMS and curing agent are mixed at a mass ratio of 10:1, and then poured onto the multi-microchannel positive mold. After removing air bubbles by vacuuming, the mold is placed in an oven and dried at 95°C for 30 minutes. The cured PDMS is then peeled off from the multi-microchannel positive mold to obtain the PDMS chip semi-finished product.

[0027] In step three above, a capture module and an analysis module are arranged on the PDMS chip semi-finished product prepared in step two, and the PDMS chip semi-finished product is bonded to a glass slide to obtain the finished PDMS chip. The arrangement of the capture and analysis modules specifically involves: depositing electrochemical detection electrodes as electrosignal detection sub-units on the multi-microchannel surface of the PDMS chip semi-finished product. The electrochemical detection electrodes include a working electrode, a counter electrode, and a reference electrode. A responsive DNA nanostructure is immobilized on the surface of the electrochemical detection electrode to form a capture unit. The responsive DNA nanostructure incorporates a fluorescent probe, specifically modified with thiol groups. DNA nanostructures are created by dropping a thiol-modified DNA nanostructure solution onto the surface of the working electrode, thus fixing the responsive DNA nanostructure onto the working electrode. Electromagnets are then mounted on the chip to form a capture enhancement unit. The capture enhancement unit is electrically connected to the control unit, the electrical signal detection subunit is electrically connected to the electrochemical analysis unit, the fluorescence signal detection subunit is electrically connected to the image analysis unit, and both the image analysis unit and the electrochemical analysis unit are data-connected to the data interpretation unit. Finally, the data interpretation unit is data-connected to the result output unit, thereby completing the arrangement of the capture and analysis modules.

[0028] In step four above, the sample containing exosomes is pretreated to remove impurities and cell debris from the sample.

[0029] In step five above, the pretreated sample from step four is injected into the PDMS chip product from step three. Exosomes are captured by the capture module, and the exosome information is collected by the signal detection unit and sent to the analysis module. Specifically, the capture unit captures exosomes in the sample, while the control unit controls the magnetic field strength and direction of the capture enhancement unit to accelerate the capture process. The signal detection unit uses the electrical signal detection subunit to collect electrical signals and sends them to the electrochemical analysis unit, and uses the fluorescence signal detection subunit to collect fluorescence images and send them to the image analysis unit. Specifically, the capture unit captures exosomes in the sample by: the responsive DNA nanostructure specifically binds to the marker on the surface of the exosome, exciting the fluorescent probe to generate a fluorescence signal, while the electrochemical detection electrode collects the electrical signal.

[0030] In step six above, the analysis module analyzes the exosome information and outputs the analysis results. Specifically, the image analysis unit preprocesses the detected fluorescence image using image analysis algorithms, then analyzes and extracts fluorescence intensity and fluorescence distribution information, and sends the results to the data interpretation unit. The electrochemical analysis unit filters the detected electrical signal, then analyzes and extracts current information, and sends the results to the data interpretation unit. The data interpretation unit integrates current information, fluorescence intensity, and fluorescence distribution information to interpret the biomarker expression level of exosomes. The result output unit outputs the interpretation results in the form of charts.

[0031] Based on the above, the advantages of this invention are as follows: When using this invention, a PDMS (polydimethylsiloxane) chip semi-finished product with multiple microchannels is fabricated using microfluidic chip technology. By arranging a capture module and an analysis module on the PDMS chip semi-finished product, a detection system is established to obtain the PDMS chip finished product. The capture unit of the capture module uses a responsive DNA nanostructure to capture exosomes and uses a magnetic field provided by the capture enhancement unit to accelerate the capture process. The signal detection unit acquires fluorescence signal images and electrical signals. The analysis module uses an electrochemical analysis unit to extract current information from the electrical signals and an image analysis unit to extract fluorescence intensity and fluorescence distribution information from the fluorescence signal images. Then, the data interpretation unit integrates multiple information to interpret the biomarker expression level of exosomes, and the result output unit provides an intuitive output of the interpretation results. This invention not only has low detection cost, low sample consumption, and low operation difficulty, but also fast detection speed and high result reliability, which can meet the needs of large-scale sample detection.

[0032] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. An integrated method for exosome capture and analysis based on responsive DNA nanostructures, comprising the following steps: Step 1: Constructing the detection system; Step 2: Manufacturing the PDMS chip; Step 3: Deploying the detection system; Step 4: Sample pretreatment; Step 5: Exosome capture; Step 6: Exosome analysis; Its features are: In step one above, the constructed detection system includes a capture module and an analysis module. The capture module includes a capture unit, a capture enhancement unit, a control unit, and a signal detection unit. The analysis module includes an electrochemical analysis unit, an image analysis unit, a data interpretation unit, and a result output unit. The signal detection unit includes an electrical signal detection subunit and a fluorescence signal detection subunit. In step two above, a multi-microchannel positive mold is fabricated using photolithography, and then a PDMS chip semi-finished product is fabricated using the multi-microchannel positive mold. In step three above, a capture module and an analysis module are arranged on the PDMS chip semi-finished product made in step two, and the PDMS chip semi-finished product is bonded to a glass plate to obtain the PDMS chip finished product. In step four above, the sample containing exosomes is pretreated to remove impurities and cell debris from the sample. In step five above, the preprocessed sample from step four is injected into the PDMS chip product from step three. The exosomes are captured by the capture module, and the exosome information is collected by the signal detection unit and sent to the analysis module. In step six above, the analysis module analyzes the exosome information and outputs the analysis results.

2. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 1, characterized in that: In step two, the process of fabricating a multi-microchannel template using photolithography involves: spin-coating photoresist onto a silicon wafer, pre-baking it, placing it in a photolithography machine, covering it with a mask and exposing it, and then post-baking it and developing it with a developer to obtain a multi-microchannel positive mold.

3. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 1, characterized in that: In step two, the process of fabricating a PDMS chip semi-finished product using a multi-microchannel template is as follows: PDMS and curing agent are mixed at a mass ratio of 10:1, then poured onto a multi-microchannel positive mold. After removing air bubbles by vacuuming, the mixture is placed in an oven and dried at 95°C for 30 minutes. The cured PDMS is then peeled off from the multi-microchannel positive mold to obtain the PDMS chip semi-finished product.

4. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 1, characterized in that: In step three, the arrangement of the capture module and the analysis module specifically involves: depositing electrochemical detection electrodes on the multi-microchannel surface of the PDMS chip semi-finished product as an electrochemical signal detection subunit; fixing responsive DNA nanostructures on the surface of the electrochemical detection electrodes to form a capture unit; installing an electromagnet on the chip to form a capture enhancement unit; establishing an electrical connection between the capture enhancement unit and the control unit; establishing an electrical connection between the electrochemical signal detection subunit and the electrochemical analysis unit; establishing an electrical connection between the fluorescence signal detection subunit and the image analysis unit; establishing a data connection between both the image analysis unit and the electrochemical analysis unit and the data interpretation unit; and establishing a data connection between the data interpretation unit and the result output unit, thereby completing the arrangement of the capture module and the analysis module.

5. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 4, characterized in that: The responsive DNA nanostructure contains a fluorescent probe.

6. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 4, characterized in that: The electrochemical detection electrode includes a working electrode, a counter electrode, and a reference electrode.

7. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 4, characterized in that: The specific method for immobilizing responsive DNA nanostructures on the surface of an electrochemical detection electrode is as follows: the responsive DNA nanostructures are modified with thiol groups, and the thiol-modified DNA nanostructure solution is dropped onto the surface of the working electrode, thereby immobilizing the responsive DNA nanostructures on the working electrode.

8. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 1, characterized in that: In step five, the capture module captures exosomes, and the signal detection unit collects exosome information for the analysis module. Specifically, the capture module captures exosomes in the sample, while the control unit controls the magnetic field strength and direction of the capture enhancement unit to accelerate the capture process. The signal detection unit collects electrical signals using the electrical signal detection subunit and sends them to the electrochemical analysis unit, and collects fluorescence images using the fluorescence signal detection subunit and sends them to the image analysis unit.

9. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 8, characterized in that: The capture unit captures exosomes in the sample by specifically binding a responsive DNA nanostructure to a marker on the surface of the exosome, exciting a fluorescent probe to generate a fluorescent signal, while an electrochemical detection electrode collects an electrical signal.

10. The integrated method for exosome capture and analysis based on responsive DNA nanostructures according to claim 1, characterized in that: In step six, the analysis module analyzes the exosome information and outputs the analysis results as follows: the image analysis unit preprocesses the detected fluorescence image using an image analysis algorithm, then analyzes and extracts fluorescence intensity and fluorescence distribution information, and sends the results to the data interpretation unit; the electrochemical analysis unit filters the detected electrical signal, then analyzes and extracts current information, and sends the results to the data interpretation unit; the data interpretation unit integrates current information, fluorescence intensity, and fluorescence distribution information to interpret the biomarker expression level of exosomes; and the result output unit outputs the interpretation results in the form of charts.

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