Water eDNA filter, online collection and solid phase extraction enrichment integrated device, method and application

Abstract

The present application relates to water body eDNA filter, online collection and solid phase extraction enrichment integrated device, method and application, and belongs to the technical field of environmental equipment. The filter comprises detachably and sealingly connected filter upper shell and lower shell, the filter upper shell or / and the lower shell is provided with a filter plate, and the filter upper shell and the lower shell are respectively provided with a water inlet and a water outlet; the filter lower shell is further provided with a counterweight. The integrated device comprises the water body eDNA filter and is connected with the water collector through the communication pipe, the water collector is connected with the negative pressure pump and the solid phase extraction instrument through the communication pipe, and the negative pressure pump is further connected with the power supply. The water body eDNA filter can realize in-situ filtering and collection of water body eDNA. The water body eDNA online collection and solid phase extraction enrichment integrated device realizes the synchronous completion of water body eDNA capture and dissolved matter enrichment in the field through the integrated fluid path design.

Description

Technical Field

[0001] This invention relates to the field of environmental DNA detection equipment technology, specifically to a water body eDNA filter and integrated collection and enrichment device for aquatic ecological monitoring, and particularly to an integrated field monitoring device for in-situ suspended eDNA filtration and simultaneous enrichment of dissolved organic matter. Background Technology

[0002] In the fields of environmental DNA (eDNA) detection and water quality analysis, existing technologies typically employ a separate operational process: eDNA samples must be collected and filtered on-site using an independent membrane filter before being cryogenically stored and transported to the laboratory for DNA extraction and PCR analysis; while the detection of target organic matter relies on laboratory solid-phase extraction (SPE) equipment for offline enrichment of frozen water samples. This discrete technical system has significant limitations—sample processing cycles can take several days to weeks, eDNA is prone to degradation during transport, and the need for repeated sampling in both processes doubles the labor and time costs. Although some portable filtration devices or small SPE devices exist on the market, they are limited to single-function modules and lack system integration design, failing to simultaneously meet the combined needs of molecular biology and chemical analysis.

[0003] Meanwhile, existing outdoor equipment generally suffers from low automation, inability to autonomously control flow rate and pressure, lack of in-situ pollution prevention mechanisms, and insufficient environmental adaptability, making it difficult to achieve high-throughput, high-fidelity in-situ rapid analysis, severely restricting the timeliness and data correlation of aquatic ecological monitoring. The main technical shortcomings are as follows: First, the discrete operating system leads to low efficiency and cost redundancy. eDNA filtration and solid-phase extraction require separate equipment, and repeated sampling processes increase time costs, while double the cost of consumables pushes up monitoring costs. Second, sample processing timeliness is poor. Traditional offline processes rely on laboratory cryopreservation (-20℃) and manual transport, resulting in a high eDNA degradation rate, and target organic matter is easily affected by photolysis or volatilization due to lack of in-situ enrichment. Furthermore, in terms of sampling methods, existing equipment generally relies on pump suction or extended pipelines to extract water samples, failing to achieve true in-situ sampling. Excessively long sampling pipelines not only easily attract impurities and contaminate subsequent samples, but their aspiration process can also disrupt the in-situ biological community. Furthermore, long-distance transport may alter the physicochemical properties of water samples, ultimately leading to a significant reduction in sample representativeness. Thirdly, the equipment integration is insufficient. Existing portable membrane filter devices mostly employ a single mechanical filtration structure (such as a manual pump or gravity drive), lacking a flow path coupling design with the SPE column module, resulting in a bulky system and insufficient flow rate control precision. These shortcomings collectively restrict the large-scale application of multimodal in-situ water monitoring technology.

[0004] Furthermore, CN119413519A discloses an in-situ biological exposure and pollutant-environmental DNA co-sampling system. This system involves setting up a reservoir on shore and using multiple pumps to send water samples separately into an extraction column (for pollutant enrichment) and an environmental DNA sampler. Its advantage is the ability to co-monitor pollutants and eDNA, but it relies on fixed facilities such as reservoirs, has long sampling pipelines, and cannot truly achieve in-situ water sampling. eDNA water samples need to be pumped to the shore-based device, during which the in-situ biological community may be disturbed, reducing sample representativeness. In contrast, the filter of this invention is placed directly in the target water layer for filtration, eliminating the need for long pipelines and intermediate reservoirs, truly achieving in-situ suspended sampling, and avoiding the problems of excessively long sampling lines and non-in-situ sampling present in CN119413519A. While CN119413519A incorporates the idea of ​​parallel pollutant extraction and eDNA sampling, its device is bulky, inconvenient, and modular; this invention organically combines filtration and solid-phase extraction into an integrated portable device, achieving a higher degree of functional integration.

[0005] CN114993765A discloses a large-scale zooplankton environmental DNA field sample collection device. This device uses a vacuum pump and a retractable sampling tube to extract water samples from the water body to a ground-based container for filtration. Its drawback is that it requires a long sampling pipeline to transfer the water sample to a filter or container, which is not true in-situ filtration; during field operation, the pipeline is prone to introducing impurities and contamination, and the pumping process may disturb the aquatic environment. The filter of this invention is suspended in the water by its own buoyancy and counterweight, eliminating the need for an external long-tube pumping system, achieving on-site sampling and filtration, and avoiding contamination and property changes caused by long-distance water sample transport. Furthermore, CN114993765A does not involve any solid-phase extraction module, only capable of obtaining eDNA samples; while this invention, while filtering eDNA, directly sends the filtered water into an SPE column to enrich organic matter, achieving simultaneous dual-target collection, making it more comprehensive.

[0006] CN118360143A discloses a system integrating a collection unit and an enrichment unit for collecting pore water and enriching benthic environmental DNA by inserting it into bottom sediment. It expands the means of in-situ online automated monitoring, demonstrating existing explorations in the field enrichment of environmental DNA. However, this device focuses on sampling sediment permeate, targeting only biological eDNA, and uses pipelines to pump the permeate to an above-ground enrichment unit. In contrast, this invention focuses on in-situ filtration of eDNA in water (water column) and innovatively integrates water quality chemical analysis (SPE enrichment of dissolved organic matter) into the same device. CN118360143A does not address filter replacement or anti-fouling design, nor does it handle chemical pollutants; therefore, it differs significantly from this invention in terms of functional integration and scope of application.

[0007] CN118562598A discloses a contamination-resistant environmental DNA sampling device and method. The device includes a vacuum chamber, a disposable filter, and a control valve, and can operate in multiple modes such as vacuuming, suction filtration, underwater in-situ sampling, and venting. It achieves underwater in-situ filtration sampling of environmental DNA and avoids cross-contamination through a disposable filter membrane assembly. In comparison, this invention is similar in that both improve the ease of filter membrane replacement: CN118562598A uses a replaceable disposable filter membrane assembly (with threaded upper and lower shells connecting the filter housing and an internal filter membrane), while the filter of this invention is designed with upper and lower shells clamping and sealing the filter membrane, allowing for quick disassembly and replacement. Both share a similar concept, aiming to reduce the risk of contamination during filter membrane replacement. However, CN118562598A remains a single eDNA filtration device and does not integrate a solid-phase extraction module, thus lacking the ability to simultaneously enrich organic matter. Furthermore, the filter of this invention features buoyancy in the upper housing and counterweight in the lower housing, allowing it to autonomously suspend itself in the target water layer for sampling; while CN118562598A primarily relies on a vacuum chamber for pressurization and pumping, and its specification does not mention the design of using buoyancy / counterweight to maintain the device's attitude. In summary, CN118562598A solves the problem of cross-contamination from repeated eDNA sampling in the field, but lacks innovative features such as simultaneous "dual-target" sampling and in-situ suspension with buoyancy and counterweight.

[0008] In summary, existing technologies generally employ pump-suction or pipeline-based sampling methods, which prevent true in-situ filtration sampling. Furthermore, traditional devices are cumbersome and prone to contamination when replacing filter membranes. While improvements such as disposable filter heads and dedicated filter membrane carriers have been proposed, these solutions mostly focus on single-module functions and lack system integration, failing to simultaneously meet the combined needs of environmental DNA collection and organic matter enrichment. Summary of the Invention

[0009] In view of the above-mentioned state of the prior art, one object of the present invention is to provide an aquatic eDNA filter that adopts a buoyancy-counterweight synergistic mechanism to achieve in-situ suspension filtration and sampling of aquatic eDNA in a designated water layer, avoiding the impact on in-situ organisms caused by excessively long sampling lines of the device; at the same time, the filter adopts an upper and lower sealed clamp structure design, which makes the filter membrane replacement process quick and convenient, and minimizes the risk of cross-contamination of samples.

[0010] The second objective of this invention is to provide an integrated device for online collection and solid-phase extraction enrichment of eDNA in water. This portable device integrates filtration, sample collection, and solid-phase extraction enrichment functions, enabling in-situ simultaneous collection of two types of samples: eDNA biological targets and dissolved organic chemical targets. This simultaneous collection of biological and chemical "dual targets" improves the field sampling efficiency, sample quality, and data reliability for eDNA and pollutant monitoring.

[0011] The third objective of this invention is to provide an integrated method for online collection and solid-phase extraction enrichment of eDNA in water.

[0012] The fourth objective of this invention is to provide an integrated device for online collection and solid-phase extraction enrichment of eDNA in water.

[0013] The technical solutions for achieving the above objectives can be summarized as follows: The water eDNA filter includes a detachably sealed upper filter shell and a lower filter shell. A filter plate is installed inside the upper filter shell and / or the lower filter shell. The upper filter shell and the lower filter shell are respectively provided with an inlet and an outlet. The lower filter shell is also provided with a counterweight so that the center of gravity of the water eDNA filter is located in the area of ​​the lower filter shell, which is used for the vertical suspension of the water eDNA filter in the water.

[0014] According to the present invention, preferably, a fixing plate with a porous structure is attached above the filter plate; more preferably, the fixing plate is a porous ceramic plate, most preferably a porous alumina ceramic plate; the pore size of the fixing plate is 10μm-100μm, more preferably 20μm-80μm.

[0015] According to the present invention, preferably, the filter plate is a mesh frame structure, more preferably a stainless steel woven mesh frame; the pore size of the filter plate is 0.1mm-5mm, more preferably 0.2mm-2mm.

[0016] According to the present invention, preferably, the upper filter housing and the lower filter housing are connected by a threaded seal; more preferably, a sealing ring is also provided at the connection between the upper filter housing and the lower filter housing.

[0017] According to the present invention, preferably, the upper and lower filter shells are hemispherical structures.

[0018] According to the present invention, preferably, the counterweight is a disc structure with a central hole, and the water outlet passes through the center of the disc structure.

[0019] According to the present invention, an integrated device for online collection and solid-phase extraction enrichment of water eDNA is also provided, comprising the above-mentioned water eDNA filter, wherein the water eDNA filter is connected to a water collector through a connecting pipe, the water collector is connected to a negative pressure pump and a solid-phase extractor through a connecting pipe, the negative pressure pump and the solid-phase extractor are also connected through a connecting pipe, and the negative pressure pump is also connected to a power source.

[0020] According to the present invention, preferably, the water collector is a light-proof container, and more preferably a brown polycarbonate bottle; Preferably, the water collector is equipped with a liquid level sensor, which is connected to the negative pressure pump circuit; more preferably, the liquid level sensor is also connected to the alarm device circuit, and the liquid level sensor is used to trigger the negative pressure pump to stop and the alarm device to sound when the water collector is full.

[0021] According to the present invention, preferably, the power source is a battery pack, and more preferably a lithium polymer battery pack.

[0022] According to the present invention, preferably, the solid phase extraction instrument is a dual-channel solid phase extraction instrument.

[0023] According to the present invention, preferably, the connecting tube is a silicone flexible tube.

[0024] According to the present invention, preferably, the integrated device for online collection and solid-phase extraction enrichment of water eDNA further includes an integrated portable cart, in which the power supply, negative pressure pump, water collector and solid-phase extractor are mounted.

[0025] According to the present invention, an integrated method for online acquisition and solid-phase extraction enrichment of water eDNA is also provided, comprising the following steps: The water is forced through a water eDNA filter by a negative pressure pump, and the filter membrane rich in eDNA is directly obtained. The filtered water then enters a water collector, and the solid phase extractor is then pressurized to allow the water to enter the solid phase extractor, where the substances in the water are enriched into the column of the solid phase extractor.

[0026] According to the present invention, preferably, the liquid level sensor of the water collector detects the liquid level. When the water collector is full, the liquid level sensor is triggered, the negative pressure pump stops, the alarm device sounds, and solid phase extraction enrichment begins.

[0027] According to the present invention, the integrated device for online collection and solid-phase extraction enrichment of eDNA in water is also provided for use in river ecological monitoring, lake water quality and biodiversity surveys, marine biodiversity surveys and / or aquaculture environment monitoring.

[0028] The beneficial and innovative effects of this invention are as follows: 1. In-situ Suspension Sampling and Filtration: The water eDNA filter of this invention employs a buoyancy-counterweight synergistic mechanism, enabling the device to be vertically suspended in a designated water layer for filtration and sampling. This design allows the sampling process to be completed in situ, avoiding the secondary impact on water samples and interference with the in-situ ecosystem caused by pumping water through long pipelines. A review of existing technologies reveals that most previous solutions used pumps / vacuum to draw water samples into onshore or shipboard filtration devices (such as CN119413519A and CN114993765A); even those claiming "underwater in-situ sampling," such as CN118562598A, do not have freely suspended devices in the target water layer; instead, they are devices with vacuum chambers that need to be fixed in place or submerged in the water for pumping. No existing literature reports this design, which utilizes its own buoyancy and counterweight balance to achieve in-situ suspension filtration of the filter membrane in the target water layer. Therefore, this in-situ suspension sampling feature is novel. It effectively solves the problem of reduced sample representativeness caused by long-distance sampling, ensuring high fidelity in water sample collection. From a creative standpoint, combining a buoyancy module with a filtration unit is a non-obvious improvement: while those skilled in the art might consider adding weights to underwater devices to make them sink, few existing solutions utilize buoyancy elements to stabilize sampling depth. This invention uses an upper shell to provide buoyancy, while a lower shell with weights works in conjunction to maintain a specific depth, ensuring a stable and efficient sampling process. This combination significantly improves the convenience and accuracy of in-situ monitoring, representing a significant advancement over existing technologies.

[0029] 2. Rapid Membrane Replacement and Anti-Contamination Design: The filter of this invention adopts a detachable upper and lower shell clamping structure, with an internal filter plate holding the filter membrane and a sealing ring to ensure no leakage. This design makes the installation and replacement of the filter membrane very quick: simply unscrew the shell to remove the filter membrane, and after placing the new filter membrane, quickly seal it for reuse. This structure minimizes the risk of cross-contamination of samples, which is crucial for high-frequency, multi-site sampling. Several existing patents also focus on the issue of filter membrane replacement. For example, CN217628360U from the Institute of Hydrobiology, Chinese Academy of Sciences, provides a filter membrane loading structure that makes filter membrane replacement easier through specific clamping rings and support plates, reducing human error and contamination; CN118562598A uses disposable filter cartridges, allowing direct replacement of the filter membrane assembly after each sampling to avoid contamination of subsequent samples by residual eDNA from the previous sampling. Therefore, rapid filter membrane replacement and anti-contamination are not entirely new concepts. However, this invention cleverly integrates this design into a portable, all-in-one field device: the filter assembly supports quick on-site installation and removal while ensuring a tight seal with the subsequent water collection / SPE module. This dual sealing method, combining threaded seals on the upper and lower shells with O-rings, ensures convenient filter membrane replacement while guaranteeing that water samples can only enter the system through the filter membrane. Compared to the traditional method of manually removing the filter head and replacing the filter membrane with tweezers, the operation is simpler and the risk of contamination is significantly reduced. Therefore, although there are precedents for quick filter membrane replacement designs, this invention, by combining leak-proof sealing and system integration, achieves a more efficient and reliable filter membrane replacement solution, representing an improvement and significant advancement over existing technologies.

[0030] 3. Simultaneous Enrichment of Both eDNA and Organic Matter: The integrated online eDNA collection and solid-phase extraction enrichment device for water bodies of this invention can simultaneously collect biological targets (eDNA) and chemical targets (dissolved organic matter) in water bodies. This "dual-target" sampling method significantly improves the efficiency and reliability of field monitoring because it obtains molecular biological and organic chemical information from the same water sample simultaneously. In the prior art, CN119413519A attempted to deploy pollutant extraction columns and environmental DNA samplers in parallel to achieve synergistic monitoring of chemical and ecological aspects. However, as analyzed above, this approach requires the use of a reservoir and multiple pumps to send water samples into different modules, effectively resulting in two concurrent sampling channels rather than simultaneous enrichment along a single path. The device is complex and not portable. Furthermore, while portable filter membrane devices or small SPE devices exist on the market, they operate independently and lack a system integration design with flow path coupling, making it impossible to simultaneously collect both types of samples in a single field operation. This invention innovatively connects these two functional modules in series via a connecting pipeline: driven by a negative pressure pump, water samples pass sequentially through a filter membrane and an SPE column, achieving dual enrichment in a single startup without the need for repeated sampling and processing. This integrated fluid path design ensures that eDNA and organic matter originate from water samples taken at the same time and location, resulting in highly correlated and synchronous data—a technical effect difficult to achieve with previous separate processes. Given that no existing single device can simultaneously produce filter membrane samples for molecular detection and SPE column samples for chemical analysis, the "dual-target simultaneous enrichment" feature of this invention possesses outstanding novelty and inventiveness. It significantly shortens the on-site sample processing cycle and reduces the time / labor costs and sample loss caused by separate sampling, thereby significantly improving monitoring efficiency and result reliability.

[0031] In summary, the key technical features of this invention, such as in-situ suspension sampling, rapid filter membrane replacement for pollution prevention, and simultaneous dual-target enrichment, are not presented in the same way in existing published patents. Each feature provides an effective solution to long-standing technical pain points, demonstrating novelty and significant progress in the field of aquatic ecological monitoring equipment. In particular, the organic combination of these features produces a synergistic effect—for example, in-situ simultaneous dual-target sampling without long pipelines not only improves the eDNA preservation rate but also ensures the integrity of chemical analysis samples—this leap in overall performance is not achieved by existing technologies. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the main structure of the water eDNA filter of the present invention.

[0033] Figure 2 This is a schematic diagram of the upper and lower shells of the water eDNA filter of the present invention.

[0034] Figure 3This is a schematic diagram of the structure of the water eDNA filter plate of the present invention.

[0035] Figure 4 This is a schematic diagram of the structure of the water body eDNA filter fixing plate of the present invention.

[0036] Figure 5 This is a schematic diagram of the sealing ring of the water eDNA filter of the present invention.

[0037] Figure 6 This is a schematic diagram of the structure of the counterweight block of the water body eDNA filter of the present invention.

[0038] Figure 7 This is a schematic diagram of the main structure of the integrated device for online collection and solid-phase extraction enrichment of eDNA in water according to the present invention.

[0039] Figure 8 This is a three-dimensional structural diagram of the upper shell, lower shell, filter plate, fixing plate, sealing ring, and counterweight of the water eDNA filter of the present invention.

[0040] Figure 9 This is a flowchart of the integrated method for online collection and solid-phase extraction enrichment of eDNA in water according to the present invention.

[0041] The components include: 1. Water eDNA filter, 101. Filter upper shell, 102. Filter lower shell, 103. Sealing ring, 104. Filter plate, 105. Fixing plate, 106. Counterweight, 107. Inlet, 108. Outlet, 2. Power supply, 3. Negative pressure pump, 4. Connecting pipe, 5. Water collector, 6. Solid phase extractor, 7. Portable cart. Detailed Implementation

[0042] This invention provides a water eDNA filter that enables in-situ sampling and filtration of water eDNA, avoiding the impact on in-situ organisms caused by excessively long sampling lines. This invention also provides an integrated device for online collection and solid-phase extraction enrichment of water eDNA, enabling simultaneous in-situ capture of water eDNA and enrichment of dissolved substances in the field, improving the field sampling efficiency, sample quality, and data reliability for eDNA and pollutant monitoring.

[0043] The water eDNA filter 1 of the present invention includes a detachably and sealed upper filter shell 101 and a lower filter shell 102. A filter plate 104 is provided inside the upper filter shell 101 and / or the lower filter shell 102. The upper filter shell 101 and the lower filter shell 102 are respectively provided with an inlet 107 and an outlet 108. The lower filter shell 102 is also provided with a counterweight 106 so that the center of gravity of the water eDNA filter is located in the area of ​​the lower filter shell 102, for the water eDNA filter to be vertically suspended in the water.

[0044] In one or more preferred embodiments, a porous fixing plate 105 is attached above the filter plate 104. When collecting eDNA from water under negative pressure, the fixing plate 105 can evenly distribute the negative pressure, protect the filter plate 104, prevent the filter plate 104 from clogging and breaking, and improve the eDNA retention rate.

[0045] In one or more preferred embodiments, the fixing plate 105 is a porous ceramic plate, most preferably a porous alumina ceramic plate; the pore size of the fixing plate 105 is 10μm-100μm, more preferably 20μm-80μm.

[0046] In one or more preferred embodiments, the filter plate 104 is a mesh frame structure, more preferably a stainless steel woven mesh frame; the pore size of the filter plate 104 is 0.1mm-5mm, more preferably 0.2mm-2mm. Three mesh frame designs are available to suit different water bodies: 0.2mm-0.5mm: suitable for low-turbidity clean water bodies, with high filtration accuracy but slower flow rate; 0.5mm-1mm: balancing filtration efficiency and flow rate, suitable for conventional water bodies; 1mm-2mm: suitable for high-turbidity water bodies, with fast flow rate.

[0047] In one or more preferred embodiments, the upper filter housing 101 and the lower filter housing 102 are connected by a threaded seal; more preferably, a sealing ring 103 is also provided at the connection between the upper filter housing 101 and the lower filter housing 102. The upper filter housing 101 and the lower filter housing 102 are detachably sealed, ensuring easy replacement of the filter plate 104 without leakage. The double-sealing design ensures that water enters the system only through the filter plate 104.

[0048] In one or more preferred embodiments, the upper filter housing 101 and the lower filter housing 102 are hemispherical structures. The spherical structure formed by the upper filter housing 101 and the lower filter housing 102 is more conducive to floating and stability in water.

[0049] In one or more preferred embodiments, the counterweight 106 is a disc structure with a central hole, and the outlet 108 passes through the center of the disc structure. The lower filter housing 102 is equipped with the counterweight 106; the synergistic effect of buoyancy and the counterweight ensures the stability of the sampling depth. Under negative pressure, the upper filter housing 101 provides buoyancy, which, in conjunction with the counterweight 106, suspends the water eDNA filter in the water. Different counterweights 106 can be used to suspend the eDNA filter at different positions in the water, achieving precise filtration and sampling of a specified water layer.

[0050] The present invention also provides an integrated device for online collection and solid-phase extraction enrichment of water eDNA, including the above-mentioned water eDNA filter 1. The water eDNA filter 1 is connected to a water collector 5 through a connecting pipe 4. The water collector 5 is connected to a negative pressure pump 3 and a solid-phase extractor 6 through the connecting pipe 4. The negative pressure pump 3 and the solid-phase extractor 6 are also connected through the connecting pipe 4. The negative pressure pump 3 is also connected to a power supply 2.

[0051] In one or more preferred embodiments, the water collector 5 is a light-proof container, more preferably a brown polycarbonate bottle; the brown polycarbonate material can protect the integrity of the photosensitive material.

[0052] In one or more preferred embodiments, a liquid level sensor is installed inside the water collector 5, and the liquid level sensor is electrically connected to the negative pressure pump 3; more preferably, the liquid level sensor is also electrically connected to an alarm device, and the liquid level sensor is used to trigger the negative pressure pump 3 to stop and the alarm device to sound when the water collector 5 is full. The water collector 5 has a built-in sensor that triggers the negative pressure pump 3 to stop and a buzzer alarm to sound when it is full, which can solve the risk of overflow when unattended in the field.

[0053] In one or more preferred embodiments, the power source 2 is a battery pack, more preferably a lithium polymer battery pack. The battery pack can use high-density lithium polymer batteries, which are easy to carry, waterproof, and provide long battery life and fast charging.

[0054] In one or more preferred embodiments, the solid-phase extraction apparatus 6 is a dual-channel solid-phase extraction apparatus. Using a dual-channel solid-phase extraction apparatus allows both channels to operate simultaneously, ensuring parallel experiments.

[0055] In one or more preferred embodiments, the connecting pipe 4 is a silicone flexible tube. The connecting pipe 4 can be made of medical-grade silicone flexible tube, which is pressure-resistant and bend-resistant, and can be combined with a quick-connect Luer connector to avoid the risk of air leakage and contamination.

[0056] In one or more preferred embodiments, the integrated device for online collection and solid-phase extraction enrichment of water-borne eDNA further includes an integrated portable cart 7, in which the power supply 2, negative pressure pump 3, water collector 5, and solid-phase extractor 6 are mounted. The entire device is integrated into the portable cart 7, and combined with sealed pipelines and dual-channel solid-phase extraction for parallel enrichment, it significantly improves the field sampling efficiency, sample quality, and data reliability for eDNA and pollutant monitoring.

[0057] The engineering process of this invention: Water is pressurized by a negative pressure pump 3, forcing it through an eDNA filter 1 to directly obtain a filter membrane rich in eDNA. The filtered water then enters a water collector 5, and is subsequently pressurized by a solid-phase extraction (SPE) instrument 6, where the substances in the water are enriched onto a column. In this invention, the water first passes through a filter plate 104 to trap eDNA particles, the filtrate is temporarily stored in the water collector 5, and then driven to the SPE instrument 6 to adsorb dissolved substances, requiring no manual intervention. Placing the water near an outdoor body of water for approximately three hours simultaneously yields a filter membrane for eDNA measurement and an enriched solid-phase column for environmental mass spectrometry analysis, offering convenience and speed.

[0058] The integrated device of this invention is particularly suitable for various aquatic environmental DNA (eDNA) research and application scenarios, including but not limited to river ecological monitoring (e.g., for continuous or fixed-point collection and analysis of water bodies, tracking fish, benthic organisms and invasive species to assess ecosystem health), lake water quality and biodiversity surveys (e.g., for sampling at different depths or in different areas, analyzing plankton and microbial communities to assist in water quality assessment and eutrophication research), marine biodiversity census (e.g., for efficient acquisition of eDNA information of fish, invertebrates, mammals, etc. in nearshore, estuary or specific sea areas for resource assessment and endangered species tracking), and aquaculture environment monitoring (e.g., for regular sampling and monitoring of pathogenic microorganisms, harmful algae and aquaculture organism eDNA dynamics, disease early warning and assessment of aquaculture environment health status), providing support for efficient collection of water samples in the field and real-time enrichment and processing of eDNA.

[0059] The present invention will be further described below through specific implementations and in conjunction with the accompanying drawings, but is not limited thereto. Example 1

[0060] like Figures 1-6 As shown, a water eDNA filter includes a detachably sealed upper filter housing 101 and a lower filter housing 102. A filter plate 104 is disposed inside the upper filter housing 101. The upper filter housing 101 and the lower filter housing 102 are respectively provided with an inlet 107 and an outlet 108. The lower filter housing 102 is also provided with a counterweight 106 so that the center of gravity of the water eDNA filter is located in the area of ​​the lower filter housing 102, which is used for the water eDNA filter to be vertically suspended in the water body, and can be adapted to the water body of different depths by increasing the weight of the counterweight.

[0061] In this embodiment, the upper filter housing 101 and the lower filter housing 102 are hemispherical structures, and are connected by a threaded seal. A silicone sealing ring 103 is also provided at the connection between the upper filter housing 101 and the lower filter housing 102, allowing for disassembly and cleaning. The filter plate 104 is a stainless steel woven mesh frame with a pore size of 1mm and a diameter of 47mm. The counterweight 106 is a circular disc structure with a central hole, and the outlet 108 passes through the center of the disc structure. Example 2

[0062] As described in Example 1, the difference is: A porous fixing plate 105 is attached to the top of the filter plate 104. The fixing plate 105 is a porous alumina ceramic plate; the pore size of the fixing plate 105 is 20μm, the diameter is 47mm, and the thickness is 5mm. Example 3

[0063] As described in Example 2, the difference is: The filter plate 104 has a pore size of 0.1 mm, and the fixing plate 105 has a pore size of 10 μm. Example 4

[0064] As described in Example 2, the difference is: The filter plate 104 has a pore size of 2 mm, and the fixing plate 105 has a pore size of 80 μm. Example 5

[0065] like Figure 7 As shown, an integrated device for online collection and solid-phase extraction enrichment of water eDNA includes a water eDNA filter 1 as described in any one of Examples 1-4. The water eDNA filter 1 is connected to a water collector 5 via a connecting pipe 4. The water collector 5 is connected to a negative pressure pump 3 and a solid-phase extractor 6 via the connecting pipe 4. The negative pressure pump 3 and the solid-phase extractor 6 are also connected via the connecting pipe 4. The negative pressure pump 3 is also connected to a power supply 2.

[0066] In this embodiment, the water collector 5 is a brown polycarbonate bottle with a volume of 1000ml. The power supply 2 is a lithium polymer battery pack. The connecting pipe 4 is a medical-grade silicone tubing with a quick-connect Luer connector. The connecting pipe 4 has an inner diameter of 10mm and a single length of 1.5m. The negative pressure pump 3 is a vacuum pump driven by a miniature brushless motor, which can adjust the pumping pressure and is compatible with the power supply 2. Example 6

[0067] As described in Example 5, the difference is: The water collector 5 is equipped with a liquid level sensor, which is connected to the negative pressure pump 3 by circuit. The liquid level sensor is also connected to the alarm device by circuit. The liquid level sensor is used to trigger the negative pressure pump 3 to stop and the alarm device to sound when the water collector 5 is full. Example 7

[0068] As described in Example 6, the difference is: The solid phase extraction instrument 6 is a dual-channel solid phase extraction instrument. Example 8

[0069] As described in Example 7, the difference is: The integrated device for online collection and solid-phase extraction enrichment of eDNA in water also includes an integrated portable cart 7, in which a power supply 2, a negative pressure pump 3, a water collector 5, and a solid-phase extractor 6 are mounted. Example 9

[0070] like Figure 9 As shown, the integrated method for online acquisition and solid-phase extraction enrichment of water eDNA includes the use of the integrated device for online acquisition and solid-phase extraction enrichment of water eDNA described in Example 8, and includes the following steps: Water is forced through the eDNA filter 1 by a negative pressure pump 3, directly obtaining a filter membrane rich in eDNA. The filtered water then enters the water collector 5, and is subsequently forced into the solid-phase extractor 6 by pressure pump 3. The substances in the water are then concentrated into the column of the solid-phase extractor 6. A level sensor in the water collector 5 detects the liquid level. When the water collector 5 is full, the level sensor triggers, the negative pressure pump 3 stops, the alarm sounds, and solid-phase extraction begins. Test case

[0071] The effects of the integrated device for online collection and solid-phase extraction enrichment of water eDNA of the present invention are compared and illustrated in Table 1.

[0072] Table 1

[0073] As can be seen from the comparison in Table 1 above, the present invention significantly improves the eDNA recovery efficiency and sampling time compared to existing methods. Traditional separation methods, due to their cumbersome offline processes, achieve a final eDNA recovery rate of only about 45%, are the most time-consuming, and the samples are prone to degradation. Existing improved portable filtration devices enrich eDNA on-site, increasing efficiency to about 60%, but because they lack simultaneous collection of organic matter, they require resampling for SPE enrichment, increasing time and workload. In contrast, the integrated device of the present invention can complete the filtration of eDNA and the enrichment of dissolved organic matter from an equivalent volume of water sample within just one hour, significantly increasing the eDNA recovery rate to about 85%, while simultaneously acquiring a concentrated organic matter sample. This dual improvement in efficiency and effectiveness stems from the innovative technical solution: in-situ rapid filtration reduces eDNA degradation loss, and online SPE prevents the escape of target organic matter, achieving efficient "one-stop" sample processing. Furthermore, the device of the present invention operates fully automatically in a closed system, reducing manual intervention, saving manpower and reducing the risk of cross-contamination. These improvements have made it possible for on-site water quality monitoring to move from qualitative to quantitative and high-throughput methods, significantly advancing the practical application of environmental DNA monitoring technology.

Claims

1. A water eDNA filter, characterized in that, The filter includes a detachably sealed upper filter housing (101) and a lower filter housing (102). A filter plate (104) is provided inside the upper filter housing (101) and / or the lower filter housing (102). The upper filter housing (101) and the lower filter housing (102) are respectively provided with an inlet (107) and an outlet (108). The lower filter housing (102) is also provided with a counterweight (106) so that the center of gravity of the water eDNA filter is located in the area of ​​the lower filter housing (102), which is used for the water eDNA filter to be vertically suspended in the water.

2. The water eDNA filter according to claim 1, characterized in that, A porous fixing plate (105) is attached to the top of the filter plate (104); preferably, the fixing plate (105) is a porous ceramic plate with a pore size of 10μm-100μm.

3. The water eDNA filter according to claim 1, characterized in that, The filter plate (104) is a mesh structure, and the pore size of the filter plate (104) is 0.1mm-5mm; Preferably, the upper filter housing (101) and the lower filter housing (102) are connected by a threaded seal.

4. The water eDNA filter according to claim 1, characterized in that, The counterweight (106) is a disc structure with a hole in the center, and the water outlet (108) passes through the center of the disc structure.

5. An integrated device for online collection and solid-phase extraction enrichment of eDNA in water, characterized in that, The device includes a water eDNA filter (1) as described in any one of claims 1-4, the water eDNA filter (1) being connected to a water collector (5) via a connecting pipe (4), the water collector (5) being connected to a negative pressure pump (3) and a solid phase extractor (6) via the connecting pipe (4), the negative pressure pump (3) and the solid phase extractor (6) being connected via the connecting pipe (4), and the negative pressure pump (3) being connected to a power source (2).

6. The integrated device for online acquisition and solid-phase extraction enrichment of water-borne eDNA according to claim 5, characterized in that, The water collector (5) is equipped with a liquid level sensor, which is connected to the negative pressure pump (3) by circuit. The liquid level sensor is also connected to the alarm device by circuit. The liquid level sensor is used to trigger the negative pressure pump (3) to stop and the alarm device to sound when the water collector (5) is full.

7. The integrated device for online acquisition and solid-phase extraction enrichment of eDNA in water according to claim 6, characterized in that, The solid phase extraction instrument (6) is a dual-channel solid phase extraction instrument.

8. The integrated device for online acquisition and solid-phase extraction enrichment of water-borne eDNA according to any one of claims 5-7, characterized in that, The integrated device for online collection and solid-phase extraction enrichment of water eDNA also includes an integrated portable cart (7), a power supply (2), a negative pressure pump (3), a water collector (5), and a solid-phase extractor (6) installed in the integrated portable cart (7).

9. A method for integrated online acquisition and solid-phase extraction enrichment of water body eDNA, comprising using the integrated device for online acquisition and solid-phase extraction enrichment of water body eDNA as described in any one of claims 5-7, characterized in that, The steps include the following: The water is pressurized by the negative pressure pump (3) and filtered through the water eDNA filter (1) to directly obtain the filter membrane rich in eDNA; then the filtered water enters the water collector (5), and then the solid phase extractor (6) is pressurized to allow the water to enter the solid phase extractor (6), and the substances in the water are enriched into the column of the solid phase extractor (6). Preferably, the liquid level sensor of the water collector (5) detects the liquid level. When the water collector (5) is full, the liquid level sensor is triggered, the negative pressure pump (3) stops, the alarm device alarms, and solid phase extraction enrichment begins.

10. The use of the integrated device for online acquisition and solid-phase extraction enrichment of water-borne eDNA according to any one of claims 5-7, characterized in that, Used for river ecological monitoring, lake water quality and biodiversity surveys, marine biodiversity censuses, and / or aquaculture environment monitoring.

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