A photoelectric enrichment system applied to a liquid biopsy sample

Abstract

The application provides a photoelectric enrichment system applied to a liquid biopsy sample, and belongs to the technical field of medical detection, and comprises the following: photoelectric nanoprobes are arranged to be used for marking a to-be-detected substance in the liquid biopsy sample and moving directionally under the driving of a light field and an electric field; the photoelectric enrichment device comprises an incubation unit, a microfluidic separation unit, a photoelectric enrichment unit, a light source device, an electric field device and a transfer unit; the incubation unit, the microfluidic separation unit and the photoelectric enrichment unit are sequentially connected through pipes; the electric field device is connected with the photoelectric enrichment unit and can apply an electric field to the photoelectric enrichment unit; and the light source device is close to the photoelectric enrichment unit and can apply a light field to the photoelectric enrichment unit; the application has the beneficial effect that: the application provides a device for enriching the to-be-detected substance in the liquid biopsy sample by combining the light field and the electric field, can efficiently enrich the to-be-detected substance in the liquid biopsy sample, and therefore can detect the to-be-detected substance with high sensitivity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical detection, and relates to a photoelectric enrichment system applied to a liquid biopsy sample. BACKGROUND

[0002] The liquid biopsy concept was introduced by CTCs ten years ago and rapidly expanded to ctDNA and other tumor markers, including circulating RNA (non-coding and messenger RNA), extracellular vesicles or tumor cultured platelets, etc. The analysis of CTCs and ctDNA in blood is called "liquid biopsy", which opens up a new way for the diagnosis of cancer and has important significance for the early detection of cancer, the improvement of cancer staging, the early detection of postoperative recurrence, the real-time monitoring of treatment effect, the detection of treatment targets and drug resistance mechanism research, etc. Among them, the research on CTCs and ctDNA is very active, but the concentration of CTCs and ctDNA in peripheral blood is extremely low, which is a very severe challenge to any researcher.

[0003] At present, there are various separation and detection methods, and the separation methods are mainly based on the physical properties of CTCs, immunobiological methods and microfluidic technology, including microporous filter membranes, gradient density centrifugation, electrostatic electric field, immunomagnetic beads and lateral fluidic separation methods, but there is lack of a system or device specially used for liquid biopsy sample enrichment, and CTCs in the liquid biopsy sample cannot be efficiently enriched, so CTCs cannot be detected with high sensitivity. SUMMARY

[0004] The purpose of the present application is to solve the above-mentioned problems existing in the prior art, and a photoelectric enrichment system applied to a liquid biopsy sample is provided.

[0005] The purpose of the present application can be achieved by the following technical scheme: a photoelectric enrichment system applied to a liquid biopsy sample, comprising:

[0006] Photoelectric nanoprobes, the photoelectric nanoprobes are arranged to label the analyte in the liquid biopsy sample and move directionally under the driving of a light field and an electric field;

[0007] The photoelectric enrichment device comprises an incubation unit, a microfluidic separation unit, a photoelectric enrichment unit, a light source device, an electric field device and a transport unit. The incubation unit, the microfluidic separation unit and the photoelectric enrichment unit are connected in sequence by a pipe. The transport unit is installed on the pipe and is arranged to drive the flow of liquid among the incubation unit, the microfluidic separation unit and the photoelectric enrichment unit. The electric field device is connected with the photoelectric enrichment unit and can apply an electric field to the photoelectric enrichment unit. The light source device is close to the photoelectric enrichment unit and can apply a light field to the photoelectric enrichment unit. The photoelectric enrichment unit enriches the target substance labeled by the photoelectric nanoprobes in the liquid biopsy sample through the light field and the electric field.

[0008] Preferably, the photoelectric nanoprobes comprise photoelectric nanoparticles that can be driven by the light field and the electric field, and target molecules that specifically interact with the target substance, the target molecules being located on the outer layer of the photoelectric nanoparticles.

[0009] Preferably, the incubation unit is arranged as an incubation container installed with a stirring and shaking device, and the incubation container promotes the combination of the target substance in the liquid biopsy sample with the photoelectric nanoprobes through the stirring and shaking device.

[0010] Preferably, the microfluidic separation unit is arranged as a microfluidic filter, and the transport unit drives the liquid biopsy sample to pass through the microfluidic filter at a preset flow rate to perform preliminary separation.

[0011] Preferably, the photoelectric enrichment unit comprises an enrichment container, and an electrophoretic medium is arranged in the enrichment container.

[0012] Preferably, the electric field device is arranged as an alternating current output device for providing an alternating current electric field, and the positive and negative electrodes of the alternating current output device are respectively electrically connected with the two sides of the electrophoretic medium.

[0013] Preferably, the light source device is arranged as a light emitting element of a single-wavelength light source or a mixed-wavelength light source that can emit visible light and near-infrared light, and the light emitting element is rotatably close to the enrichment container and can adjust the angle of light with the enrichment container.

[0014] Preferably, when the photoelectric enrichment unit enriches the target substance, the liquid biopsy sample after preliminary separation flows into the enrichment container, the alternating current output device applies an alternating current electric field with a voltage value of 0.1-100V, a current value of 0.001-15A and a frequency of 0.001-50kHz to the electrophoretic medium, and the light emitting element applies a light field with a wavelength of 400-2500nm, a light power density of 0.001-10W / cm 2 and a light angle of 0-180° to the enrichment container.

[0015] Preferably, the transport unit comprises at least two peristaltic pumps, and the two peristaltic pumps are respectively arranged on the pipe between the incubation unit and the microfluidic separation unit and on the pipe between the microfluidic separation unit and the optoelectronic enrichment unit.

[0016] Preferably, the liquid biopsy sample is one or more of blood, peritoneal fluid, urine, cerebrospinal fluid, bone marrow, saliva and sputum, and the analyte is one or more of circulating tumor cells, circulating DNA, exosomes, polypeptides and proteins.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] 1. The present application provides a device for enriching an analyte in a liquid biopsy sample by combining an optical field with an electric field, which can efficiently enrich the analyte in the liquid biopsy sample, and thus can sensitively detect the analyte.

[0019] 2. Compared with the prior art of enriching an analyte by using a single field (e.g. a magnetic field), the present embodiment ingeniously uses a composite field formed by an electric field and an optical field to enrich the analyte, which can greatly improve the enrichment effect and shorten the time required for enriching the analyte.

[0020] 3. Since the analyte labeled by an optoelectronic nanoprober is driven to move in a directional manner by the electric field and the optical field to achieve the enrichment effect, the subsequent separation and washing are very convenient, and the electric field and the optical field are respectively applied by an alternating current output device and a light-emitting element, so it is very convenient to remove the electric field and the optical field, and thus the device has the advantage of being easy to control the optical field and the electric field.

[0021] 4. The optoelectronic enrichment and detection system further subdivides and integrates the enrichment process, and integrates incubation, preliminary separation and enrichment together to improve the efficiency of sample processing, which not only improves the separation and enrichment effect, but also greatly improves the efficiency of sample processing.

[0022] 5. The system has a high degree of automation, and the transport unit automatically transports the liquid biopsy sample to the incubation unit, the microfluidic separation unit and the enrichment unit according to the program setting. In this process, the incubation unit, the microfluidic separation unit and the enrichment unit are driven to perform multi-stage separation and enrichment operations according to the pre-set program, so that the enrichment process of the liquid biopsy sample is more simplified and automated, and is easy to control. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 FIG. 1 is a structural schematic diagram of an optoelectronic enrichment system according to the present application.

[0024] Figure 2 FIG. 2 is a structural schematic diagram of an optoelectronic enrichment unit, a light source device and an electric field device according to the present application.

[0025] Figure 3 A cross-sectional view of the photoelectric enrichment unit, light source device, and electric field device of the present application.

[0026] Figure 4 A structural view of the incubation unit of the present application.

[0027] Figure 5 A structural view of the stirring and shaking device of the present application.

[0028] In the figure, 100, incubation container; 110, stirring and shaking device; 200, microfluidic filter; 300, enrichment container; 310, electrophoretic medium; 400, light-emitting element; 500, alternating current output device; 600, peristaltic pump; 700, tube. DETAILED DESCRIPTION

[0029] The following are specific embodiments of the present application and further describe the technical solutions of the present application in conjunction with the accompanying drawings, but the present application is not limited to these embodiments.

[0030] As shown in Figures 1-5 A photoelectric enrichment system applied to a liquid biopsy sample, comprising: photoelectric nanoprobes and a photoelectric enrichment device, the photoelectric nanoprobes are configured to label target substances in the liquid biopsy sample and move directionally under the driving of a light field and an electric field; the photoelectric enrichment device comprises an incubation unit, a microfluidic separation unit, a photoelectric enrichment unit, a light source device, an electric field device, and a transport unit, the incubation unit, the microfluidic separation unit, and the photoelectric enrichment unit are connected in sequence through a tube 700, the transport unit is installed on the tube 700 and is configured to drive the liquid to flow between the incubation unit, the microfluidic separation unit, and the photoelectric enrichment unit, the electric field device is connected with the photoelectric enrichment unit and can apply an electric field to the photoelectric enrichment unit, the light source device is close to the photoelectric enrichment unit and can apply a light field to the photoelectric enrichment unit, and the photoelectric enrichment unit enriches the target substances labeled by the photoelectric nanoprobes in the liquid biopsy sample through the light field and the electric field.

[0031] The optoelectronic nanoprobes can be driven to move directionally under the action of light and electric field, and have one or more signal functions such as surface-enhanced Raman scattering (SERS), fluorescence, absorbance, magnetic resonance and other signal detection functions, and can label the target to be detected through specific interaction with the target to be detected in the liquid biopsy sample, and separate under the action of light and electric field; in the actual structure, the incubation unit is connected with an inlet drainage pipe, the optoelectronic enrichment unit is connected with an outlet drainage pipe, and the inlet drainage pipe, the outlet drainage pipe and the pipe 700 are actually channels for flowing of the liquid biopsy sample, the optoelectronic nanoprobes and the cleaning liquid; the light source device and the electric field device are matched with the chamber in the optoelectronic enrichment unit, and are light and electric field control devices, and the light source device and the electric field device can provide stable voltage, current, alternating frequency and stable power output light source.

[0032] In the formula, the liquid biopsy sample is one or more of blood, peritoneal fluid, urine, cerebrospinal fluid, bone marrow, saliva and sputum, and the target to be detected is one or more of circulating tumor cells, circulating DNA, exosomes, polypeptides and proteins. When the target to be detected is the circulating tumor cells, the separation and enrichment efficiency can reach more than 95%; when the target to be detected is the circulating tumor cells, the detection sensitivity is 1 cells / mL, preferably 5-100 cells / mL.

[0033] Preferably, the transport unit includes at least two peristaltic pumps 600, and the two peristaltic pumps 600 are respectively arranged on the pipe 700 between the incubation unit and the microfluidic separation unit and on the pipe 700 between the microfluidic separation unit and the optoelectronic enrichment unit.

[0034] The liquid biopsy sample and the optoelectronic nanoprobes enter the incubation unit from the inlet drainage pipe for stirring and oscillation mixing, and after a period of incubation, the liquid biopsy sample mixed with the optoelectronic nanoprobes is driven by the transport unit to enter the microfluidic separation unit through the pipe 700 between the incubation unit and the microfluidic separation unit to preliminarily separate impurities, the liquid biopsy sample after preliminary separation is driven by the transport unit to enter the optoelectronic enrichment unit through the pipe 700 between the microfluidic separation unit and the optoelectronic enrichment unit, the light source device and the electric field device apply light and electric field to the optoelectronic enrichment unit according to a preset program, so that the target to be detected in the liquid biopsy sample is enriched together and waste liquid is discharged, then the light and electric field are removed, and then other interfering substances are removed by washing with phosphate buffered saline (PBS, pH 7.4), the target to be detected labeled by the optoelectronic nanoprobes is further separated and enriched, and finally the target to be detected is detected by using a spectroscopic technique, the sample after cleaning is collected, and the target to be detected after separation and enrichment is detected by using a spectroscopic detection technique (Raman spectroscopy, fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy or magnetic resonance detection technology).

[0035] The system has high degree of automation, and the liquid biopsy sample is automatically transported to the incubation unit, the microfluidic separation unit and the enrichment unit according to the program setting through the transfer unit. In the process, the incubation unit, the microfluidic separation unit and the enrichment unit are driven by the preset program to perform multi-stage separation and enrichment operation, so that the enrichment process of the liquid biopsy sample is more simple and automatic, and is convenient and controllable.

[0036] The device is provided for enriching the target in the liquid biopsy sample by combining the optical field and the electric field, which can efficiently enrich the target in the liquid biopsy sample, and therefore can detect the target with high sensitivity.

[0037] It should be noted that, compared with the existing single field (for example, magnetic field) enrichment method of the target, the complex field formed by the electric field and the optical field is ingeniously used to enrich the target in the embodiment, which can greatly improve the enrichment effect and shorten the time required for enriching the target.

[0038] In addition, since the target labeled by the optoelectronic nanoprobes is directionally driven to move by the electric field and the optical field to achieve the enrichment effect, it is very convenient for subsequent separation and washing. The electric field and the optical field are respectively applied by the alternating current output device 500 and the light emitting element 400, and it is very convenient to remove the electric field and the optical field, so it has the advantage of convenient control of light and electric field.

[0039] The optoelectronic nanoprobes comprise optoelectronic nanoparticles that can be directionally driven by the optical field and the electric field, and target molecules that specifically interact with the target.

[0040] The optoelectronic nanoparticles are selected from the group consisting of Janus structure nanoparticles formed by combination of noble metals and semiconductor materials, and the shape is selected from the group consisting of spherical, rod-shaped, linear, spiral, star-shaped, triangular sheet, cubic, triangular pyramid, or a combination thereof. The outer layer of the optoelectronic nanoparticles is modified with target molecules that specifically interact with the target, such as monoclonal antibodies, polypeptides, folic acid, aptamers, DNA fragments and galactosamine, etc. The size of the optoelectronic nanoprobes is 5-100000 nm.

[0041] The incubation unit is provided as an incubation container 100 installed with a stirring and oscillation device 110, and the incubation container 100 promotes the binding of the target in the liquid biopsy sample to the optoelectronic nanoprobes through the stirring and oscillation device 110.

[0042] The incubation unit is a component for incubating the liquid biopsy sample and the optoelectronic nanoprobes, the incubation container 100 is connected with the inlet drainage pipe, the stirring and shaking device 110 in the incubation container 100 has the functions of stirring and shaking at different rotating speeds, which can promote the combination of the analyte and the optoelectronic nanoprobes, and the rotating speed range of the stirring and shaking device 110 is 0-1000 rpm, and the stirring and shaking device 110 has the functions of clockwise and counterclockwise rotation.

[0043] The microfluidic separation unit is arranged as a microfluidic filter 200, and the transport unit drives the liquid biopsy sample to pass through the microfluidic filter 200 at a preset flow rate for preliminary separation.

[0044] It should be noted that the microfluidic filter 200 is a device for cell separation and sorting, which can separate cells based on cell size or deformability as biomarkers, thereby realizing separation at the molecular level.

[0045] The microfluidic filter 200 has an array with different sizes and spacings (microfluidic component size: 1-10000 cm 2 , array size: 0.5-1000 μm, array spacing: 0.5-500 μm), and the array size and spacing are adjustable.

[0046] The optoelectronic enrichment unit includes an enrichment container 300, and the enrichment container 300 is provided with an electrophoresis medium 310. The enrichment container 300 has a cavity, so the enrichment container 300 can accommodate the electrophoresis medium 310. It should be noted that the electric field device applies an electric field to the electrophoresis medium 310 to drive the analyte labeled by the optoelectronic nanoprobes to move directionally by the principle of electrophoresis. The electrophoresis medium 310 is a conductive solution or gel such as glucose salt solution, and the electrophoresis field size is 0.01-1000 cm 3 .

[0047] The electric field device is arranged as an alternating current output device 500 for providing an alternating current field, and the positive and negative electrodes of the alternating current output device 500 are respectively electrically connected with two sides of the electrophoresis medium 310.

[0048] It should be noted that the positive and negative electrodes of the alternating current output device 500 are respectively electrically connected with two sides of the electrophoresis medium 310, so that an alternating current field can be applied to the electrophoresis medium 310. The voltage range of the alternating current field is 0.1-100 V, the current range is 0.001-15 A, and the frequency range is 0.001-50 kHz.

[0049] The light source device is arranged as a light emitting element 400 of a single-wavelength light source or a mixed-wavelength light source capable of releasing visible light and near-infrared light, and the light emitting element 400 is rotatably arranged close to the enrichment container 300 and is capable of adjusting the angle of light with the enrichment container 300. The light field applied to the enrichment container 300 by the light emitting element 400 is a single-wavelength light source or a mixed-wavelength light source of visible light and near-infrared light, with a wavelength range of 400-2500 nm and a light power density of 0.001-10 W / cm 2 , and the illumination angle is adjusted within a range of 0-180°.

[0050] In the enrichment stage, under the irradiation of the electric field and the light field, the optoelectronic nanoprobes labeled on the analyte can be directionally driven, and according to the number of the optoelectronic nanoprobes labeled on the analyte, there is a certain difference in the moving speed of the analyte, and the analyte is separated by using the difference in the moving speed. The analyte in the sample preliminarily separated by the microfluidic filter 200 is further enriched and extracted by adding and removing one or more of the electric field and the light field.

[0051] When the optoelectronic enrichment unit enriches the analyte, the liquid biopsy sample preliminarily separated flows into the enrichment container 300, the alternating current output device 500 applies an alternating current field with a voltage value of 0.1-100 V, a current value of 0.001-15 A and a frequency of 0.001-50 kHz to the electrophoretic medium 310, and the light emitting element applies a light field with an illumination wavelength of 400-2500 nm, a light power density of 0.001-10 W / cm 2 and an illumination angle of 0-180° to the enrichment container.

[0052] The optoelectronic enrichment detection system further subdivides and integrates the enrichment process, integrates incubation, preliminary separation and enrichment together, and improves the efficiency of sample processing. In this way, not only the separation and enrichment effect is improved, but also the efficiency of sample processing is greatly improved.

[0053] Embodiment one:

[0054] (1) Preparation of optoelectronic nanoprobes:

[0055] Preparation of BiVO4 microspheres: 0.97 g of bismuth nitrate pentahydrate was added to 10 mL of acetic acid, ethanol and ultrapure water and dissolved. Then 0.234 g of ammonium metavanadate was dissolved in 20 mL of 5M NaOH solution, and the solution was added dropwise to the above bismuth nitrate solution to form a yellow-brown solution. After stirring for 5 min, the pH of the solution was adjusted to 4.6, and stirring was continued for 10 min. The mixed solution was transferred to a polytetrafluoroethylene inner tank and placed in a reaction kettle. The solution was heated at 80°C for 3 h, cooled to room temperature, and then centrifuged with ethanol and pure water three times (2000 rpm for 5 min). The BiVO4 microspheres were dried at 60°C for 12 h.

[0056] Preparation of BiVO4-Au: about 10 μg of BiVO4 microspheres were taken into 0.1 mL of ethanol and ultrasonicated for 10 min. The U-shaped coating method of a pipette was used to drop the microspheres on the surface of a silicon wafer, and the wafer was left to air dry. The wafer was placed in a vacuum chamber, and a layer of Au was vacuum evaporated on one half of the microspheres by electron beam. The evaporation rate was 0.5 A / s. Three rounds of co-sputtering evaporation were performed, and the thickness of the coating layer was 20 nm. A few drops of the solution were dropped on the surface of the coating layer using a pipette, and the prepared BiVO4-Au microspheres were repeatedly washed and sucked.

[0057] Preparation of SERS functional BiVO4-Au optoelectronic nanoprobes: 10 μL of 4-mercaptobenzoic acid (MBA) (1 mM) solution was added to 4 mL of BiVO4-Au solution, and the mixture was shaken at a speed of 200 rpm for 5 min. Then, 50 μL of 1.0 mg / mL BSA was added, and the mixture was shaken for another 15 min. Then, 0.2 mL of activated folic acid (40 mL of 1 mg / mL folic acid, 32.0 mg of EDC, and 19.2 mg of NHS were dissolved in PBS, and the mixture was reacted in the dark for 8 h) was added, and the mixture was shaken for 16 h. The SERS functional BiVO4-Au optoelectronic nanoprobes were prepared by centrifugation and dispersion in 5 mL of water.

[0058] (2) Incubation and enrichment separation of the optoelectronic nanoprobes and the CTCs to be detected in the blood sample

[0059] 0.2 mL of the optoelectronic nanoprobes was taken and added to 1.8 mL of PBS, and 2 mL of the blood sample of a breast cancer patient was introduced into the incubation container 100 through the inlet drainage tube for mixing and incubation, and the mixture was shaken at room temperature (200 rpm, 30 min).

[0060] The incubated mixture was introduced into the microfluidic filter 200 under the driving of the peristaltic pump 600 at a flow rate of 0.5 mL / min, and the blood sample was preliminarily separated, and the mononuclear cells (CTCs and white blood cells) were collected, and other substances were discharged into the waste liquid collection device. The mixture of the separated CTCs and white blood cells was introduced into the optoelectronic enrichment unit at a flow rate of 0.2 mL / min, and the electrophoretic medium 310 was a glucose salt solution, the voltage value was 5-10 V, the frequency was 0.5-1 kHz, the illumination wavelength was 808 nm, the optical power density was 40-100 mW / cm 2 (0.04-0.1 W / cm 2 ), the illumination angle was 30°, the illumination time was 15-20 min, and the electricity was turned on for 10 min for light-driven separation and enrichment. PBS buffer was introduced at a flow rate of 0.1 mL / min for washing, and the cells to be detected were collected.

[0061] The mixture of isolated CTCs and white blood cells is passed into the photoelectric enrichment unit at a flow rate of 0.2 mL / min, the electrophoretic medium 310 is a glucose salt solution, the voltage value is 5 V, the frequency is 1 kHz, and electrophoretic separation is performed for 10 min. PBS buffer is introduced at a flow rate of 0.1 mL / min for flushing, and the cells to be tested are collected.

[0062] (3) Raman detection of photoelectric nanoprobes labeled CTCs

[0063] The collected CTCs are placed under a Raman microscope for observation, spectral scanning and imaging, and the number and type of CTCs are determined according to the standard curve and Raman signal molecule spectrum. The detection conditions are: 785 nm laser, 200 mW, scanning time: 0.2 s, and scanning times: 3 times.

[0064] Example Two:

[0065] In the preparation of photoelectric nanoprobes with SERS function in step (1) of Example One, the Au electron beam vacuum evaporation is replaced by Ag electron beam vacuum evaporation. The specific separation method is as follows: about 10 μg of BiVO4 microspheres is added to 0.1 mL of ethanol and ultrasonicated for 10 min. The U-shaped coating method of a pipette is used to drop on the surface of a silicon wafer, and it is left to air dry. It is placed in a vacuum chamber, and a layer of Ag is plated on half of the microspheres by electron beam vacuum evaporation. The evaporation rate is Three rounds of co-sputtering evaporation are performed, and the thickness of the plated layer is 20 nm. A few drops of solution are dropped on the surface of the plated layer using a pipette, and the prepared BiVO4-Ag microspheres are repeatedly washed and sucked several times.

[0066] Example Three:

[0067] In the preparation of photoelectric nanoprobes with SERS function in step (1) of Example One, the Au electron beam vacuum evaporation is replaced by Pt electron beam vacuum evaporation. The specific separation method is as follows: about 10 μg of BiVO4 microspheres is added to 0.1 mL of ethanol and ultrasonicated for 10 min. The U-shaped coating method of a pipette is used to drop on the surface of a silicon wafer, and it is left to air dry. It is placed in a vacuum chamber, and a layer of Pt is plated on half of the microspheres by electron beam vacuum evaporation. The evaporation rate is Three rounds of co-sputtering evaporation are performed, and the thickness of the plated layer is 20 nm. A few drops of solution are dropped on the surface of the plated layer using a pipette, and the prepared BiVO4-Ag microspheres are repeatedly washed and sucked several times.

[0068] Example Four:

[0069] The target molecule in the preparation of the SERS functional BiVO4-Au photoelectric nanoprobes of the embodiment step (1) is replaced by anti-EpCAM antibody to prepare SERS functional photoelectric nanoprobes for targeting breast cancer circulating tumor cells, and the preparation method is briefly described as follows: 10 μL of 4-mercaptobenzoic acid (MBA) (1 mM) solution is added dropwise into 4 mL of BiVO4-Au solution, and oscillated at 200 rpm for 5 min, 50 μL of 1.0 mg / mL BSA is added, and oscillation is continued for 15 min. 0.2 mL of activated anti-EpCAM monoclonal antibody (10 mL of 1 mg / mL anti-EpCAM monoclonal antibody, 32.0 mg of EDC, and 19.2 mg of NHS are dissolved in PBS, and the reaction is carried out in the dark for 8 h) is added, oscillated for 16 h, centrifuged and dispersed in 5 mL of water to obtain the SERS functional BiVO4-Au photoelectric nanoprobes.

[0070] It should be noted that all the directionality indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present application are only used to explain the relative position relationship, movement condition, etc. between the components in a certain specific posture (as shown in the drawings), and if the specific posture changes, the directionality indications will also change accordingly.

[0071] In addition, the descriptions such as "first", "second", "one" and the like in the present application are only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" can be explicitly or implicitly included at least one of the features.

[0072] In the present application, unless otherwise explicitly specified and limited, the terms "connection", "fixation" and the like should be understood in a broad sense, for example, "fixation" can be fixed connection, or detachable connection, or integral; can be mechanical connection, or electrical connection; can be direct connection, or indirect connection through an intermediate medium; can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise explicitly limited.

[0073] In addition, the technical solutions of each embodiment of the present application can be combined with each other, but it must be based on the realization of a person skilled in the art, and when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor is it within the protection scope required by the present application.

Claims

1. A photoelectric enrichment system for liquid biopsy samples, characterized in that, include: An optoelectronic nanoprobe, which is configured to label analytes in liquid biopsy samples and move directionally under the drive of an optical and electric field; A photoelectric enrichment device includes an incubation unit, a microfluidic separation unit, a photoelectric enrichment unit, a light source device, an electric field device, and a transport unit. The incubation unit, the microfluidic separation unit, and the photoelectric enrichment unit are connected sequentially via tubes. The transport unit is mounted on the tubes and configured to drive liquid to flow between the incubation unit, the microfluidic separation unit, and the photoelectric enrichment unit. The electric field device is connected to the photoelectric enrichment unit and can apply an electric field to the photoelectric enrichment unit. The light source device is close to the photoelectric enrichment unit and can apply a light field to the photoelectric enrichment unit. The photoelectric enrichment unit enriches the analytes labeled by the photoelectric nanoprobes in the liquid biopsy sample through the light field and the electric field. The photoelectric nanoprobe includes photoelectric nanoparticles that can be directed by light and electric fields, and target molecules that specifically interact with the analyte, wherein the target molecules are located on the outer layer of the photoelectric nanoparticles; The photoelectric nanoparticles are selected from nanoparticles with a Janus structure formed by a combination of noble metals and semiconductor materials. The noble metal is gold, silver or platinum, and the semiconductor material is bismuth vanadate. The photoelectric nanoparticles are obtained by depositing a layer of noble metal on one half of the bismuth vanadate microspheres.

2. The photoelectric enrichment system for liquid biopsy samples as described in claim 1, characterized in that: The incubation unit is configured as an incubation container equipped with a stirring and oscillation device, which promotes the binding of the analyte in the liquid biopsy sample with the photoelectric nanoprobe through the stirring and oscillation device.

3. The photoelectric enrichment system for liquid biopsy samples as described in claim 2, characterized in that: The microfluidic separation unit is configured as a microfluidic filter, and the transport unit drives the liquid biopsy sample through the microfluidic filter at a preset flow rate for initial separation.

4. The photoelectric enrichment system for liquid biopsy samples as described in claim 3, characterized in that: The photoelectric enrichment unit includes an enrichment container, and an electrophoretic medium is disposed inside the enrichment container.

5. The photoelectric enrichment system for liquid biopsy samples as described in claim 4, characterized in that: The electric field device is configured as an AC output device for providing an alternating electric field, and the positive and negative terminals of the AC output device are electrically connected to both sides of the electrophoretic medium, respectively.

6. The photoelectric enrichment system for liquid biopsy samples as described in claim 5, characterized in that: The light source device is configured as a light-emitting element that emits a single-wavelength light source or a mixed-wavelength light source that can emit visible light and near-infrared light. The light-emitting element can be rotatably brought close to the enrichment container and the angle between the light and the enrichment container can be adjusted.

7. The photoelectric enrichment system for liquid biopsy samples as described in claim 6, characterized in that: When the photoelectric enrichment unit enriches the analyte, the liquid biopsy sample after preliminary separation flows into the enrichment container. The AC output device applies an AC electric field with a voltage of 0.1-100V, a current of 0.001-15A, and a frequency of 0.001-50kHz to the electrophoresis medium. The light-emitting element applies light to the enrichment container with a wavelength of 400-2500nm and a light power density of 0.001-10W / cm². 2 And a light field with an illumination angle of 0-180°.

8. The photoelectric enrichment system for liquid biopsy samples as described in claim 1, characterized in that: The transfer unit includes at least two peristaltic pumps, which are respectively disposed on the tube between the incubation unit and the microfluidic separation unit and on the tube between the microfluidic separation unit and the photoelectric enrichment unit.

9. The photoelectric enrichment system for liquid biopsy samples as described in claim 1, characterized in that: Liquid biopsy samples are one or more of the following: blood, ascites, urine, cerebrospinal fluid, bone marrow, saliva, and sputum. The analytes are one or more of the following: circulating tumor cells, circulating DNA, exosomes, polypeptides, and proteins.

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