Water quality biotoxicity detection chip, its preparation method and detection system

Abstract

This application provides a water quality biotoxicity detection chip, its preparation method, and a detection system. The water quality biotoxicity detection chip includes: a chip body, microchannels located inside the chip body, and multiple micropore structures. The micropore structures include: a first injection port for injecting a first liquid; a second injection port for injecting a second liquid; and a drain port for discharging liquid. The microchannels include: a mixer connected to the first injection port via a first injection pipe and connected to the second injection port via a second injection pipe; at least a partially transparent detection ring, which is connected to the reaction channel via the drain pipe and drain port, located between the mixer and the detection ring; and several reaction columns spaced apart along the extension direction of the reaction channel. Using a microfluidic detection chip for water quality biotoxicity detection based on luminescent bacteria can reduce the amount of bacterial strain used in each detection and shorten the reaction time during detection.

Description

Technical Field

[0001] This application relates to the field of wastewater treatment technology, and in particular to a water quality biotoxicity detection chip, its preparation method and detection system. Background Technology

[0002] The rapid development of modern industry has led to a continuous increase in the discharge of industrial wastewater. Most industrial wastewater contains biotoxic substances, which can severely impact wastewater treatment systems and the microorganisms in the ecosystem after being discharged into rivers, resulting in effluent exceeding standards or river pollution. Therefore, the detection and early warning of wastewater biotoxicity is an indispensable part of water environment quality monitoring.

[0003] Biological and chemical methods are the two main approaches for detecting the biotoxicity of water. However, chemical methods still have many limitations. For example, even if all types of pollutants can be measured, the combined effects of antagonistic, additive, and synergistic interactions between pollutants make it very difficult to predict the true toxicity of wastewater based solely on the concentration data of different pollutants. Therefore, chemical analysis data of water bodies cannot reflect the overall quality of the water or the impact of pollutants on individual organisms and the ecological environment.

[0004] Common biotoxicity testing methods include: acute toxicity testing of luminescent bacteria, algal cell growth inhibition testing, acute toxicity testing of daphnia and zebrafish juveniles, community-level toxicity testing, and SOS / umu genotoxicity testing. Because the acute toxicity testing of luminescent bacteria has the advantages of being rapid, economical, and requiring only a small experimental space while yielding reliable results compared to other biotoxicity tests, it is the primary method used in existing toxicity testing equipment.

[0005] However, when conducting continuous testing, toxicity detection equipment suffers from drawbacks such as requiring large quantities of bacteria, complex replacement methods when the reactor is contaminated, and long reaction times leading to untimely results. These drawbacks make the acute toxicity detection method for luminescent bacteria difficult to use in practice for continuous wastewater toxicity testing. Furthermore, the luminescent bacteria are preserved using vacuum freeze-drying. In their dormant state, the luminescence intensity rapidly decreases after the freeze-dried powder is thawed and stored at 4°C. This results in insufficient maintenance and unreliable test results during instrument use.

[0006] Therefore, existing biotoxicity detection systems based on luminescent bacteria still have some limitations. Summary of the Invention

[0007] This application provides a water quality biological toxicity detection chip, its preparation method, and detection system, aiming to solve at least some of the problems existing in the current toxicity detection system.

[0008] In a first aspect, embodiments of this application provide a water quality biotoxicity detection chip. The chip includes: a chip body, microchannels located within the chip body, and multiple micropore structures; the micropore structures include: a first injection port for injecting a first liquid; a second injection port for injecting a second liquid; and a drain port for discharging liquid; the microchannels include: a mixer connected to the first injection port via a first injection pipe and connected to the second injection port via a second injection pipe; a detection ring connected to the drain port via a drain pipe; a reaction channel located between the mixer and the detection ring; and a plurality of reaction columns; the plurality of reaction columns are spaced apart along the extension direction of the reaction channel.

[0009] Optionally, the reaction column is selected from one or more of the following: cylindrical, triangular, and square columns.

[0010] Optionally, the reaction channel length between two adjacent reaction columns is 20 μm; the diameter of each reaction column is 10 μm.

[0011] Optionally, the chip has a preset thickness; wherein, the detection ring is closer to the surface of the chip body in the thickness direction of the chip body, relative to the other microfluidic structures.

[0012] Optionally, the minimum channel width of the first injection pipe and the second injection pipe is 5 μm; the minimum channel height of the first injection pipe and the second injection pipe is 10 μm.

[0013] Optionally, the chip body is formed by bonding two sheet-like structures; the sheet-like structures are made of PDMS material; wherein the thickness of the sheet-like structures is 3-5 mm; and the area of ​​the sheet-like structures is 2-4 inches.

[0014] Secondly, embodiments of this application also provide a method for preparing the water quality biotoxicity detection chip as described above. This method includes: fabricating a chip mold based on a silicon wafer; pouring a liquid mixture into the chip mold; the liquid mixture being formed by uniformly mixing PDMS polymer monomers and a curing agent in a preset ratio; separating the cured liquid mixture from the silicon wafer to obtain a PDMS layer and drilling holes at target locations to form a microporous structure; bonding two PDMS layers to fabricate a chip body; and subjecting the chip body to at least one material modification treatment; the material modification treatment is selected from one or more of the following treatments: plasma modification; or the addition of hydrophilic groups.

[0015] Optionally, after performing at least one material modification treatment on the chip body, the method further includes: coating at least a portion of the surface of the chip body with a black coating to form a black matte coating; and before bonding the two PDMS layers, the method further includes: performing a hydrophilic treatment on the surface of the PDMS layer having a channel structure.

[0016] Thirdly, this application also provides a water quality biotoxicity detection chip system. The system includes: a reaction detection module, comprising the water quality biotoxicity detection chip and a photomultiplier tube as described above; a sample injection module for injecting reagents into the water quality biotoxicity detection chip through a first injection port and a second injection port; a reagent storage module for storing reagents; a bacterial strain storage module for storing luminescent bacteria; and a control module for controlling the sample injection by the sample injection module and generating corresponding detection results based on the detection data from the photomultiplier tube.

[0017] At least one advantage of the water quality biotoxicity detection system provided in this application is that: using a microfluidic detection chip for biotoxicity detection based on luminescent bacteria can reduce the amount of bacterial strains used in each test and shorten the reaction time. Furthermore, the chip is easily replaceable, effectively simplifying reactor replacement methods and forming a method and application device for online monitoring and early warning of biotoxicity.

[0018] Furthermore, the corridor reaction structure formed by the reaction columns in the detection chip enables rapid bacterial activation. Within the chip, PDMS material is modified using embedding and immobilization technology to increase biocompatibility and adsorb and immobilize the bacterial strains. Additionally, a black matte coating is applied to effectively detect luminescence, improving detection accuracy.

[0019] Further embodiments of the advantageous aspects of the above-described water quality biotoxicity detection system are described in detail below. All disclosures in this specification are merely exemplary, and those skilled in the art can readily make appropriate adjustments without departing from the spirit and scope of the invention as disclosed and claimed in this application. Attached Figure Description

[0020] One or more embodiments are illustrated by way of example with reference numerals in the accompanying drawings. These illustrations do not constitute a limitation on the embodiments. Elements with the same reference numerals in the drawings are denoted as similar elements. Unless otherwise stated, the figures in the drawings are not to be limited by scale.

[0021] Figure 1 A schematic diagram of the water quality biotoxicity detection chip provided in the embodiments of this application;

[0022] Figure 2A side view of the water quality biotoxicity detection chip provided in an embodiment of this application;

[0023] Figure 3 This is a schematic diagram of the structure of the reaction column provided in an embodiment of this application;

[0024] Figure 4 A flowchart of a chip fabrication method provided in the embodiments of this application;

[0025] Figure 5 This is a schematic diagram of a water quality biotoxicity detection system provided in an embodiment of this application. Detailed Implementation

[0026] To facilitate understanding of this application, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. It should be noted that when an element is described as being "fixed to" another element, it can be directly on the other element, or one or more intermediate elements may exist between them. When an element is described as being "connected" to another element, it can be directly connected to the other element, or one or more intermediate elements may exist between them. The terms "upper," "lower," "inner," "outer," "bottom," etc., used in this specification indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0027] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0028] Furthermore, the technical features involved in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0029] A "microfluidic chip" refers to a device that manipulates and processes minute amounts of liquid by using micrometer-scale channels and chambers within the chip body, combined with the effects of fluid dynamics and surface tension, to complete one or more experimental testing steps. In this application, the micrometer-scale channels and chambers are referred to as "microchannels," and the holes connecting the microchannels to the outside of the chip are referred to as "micropore structures."

[0030] Figure 1This is a schematic diagram of the microfluidic chip provided in an embodiment of this application. It can be applied to the detection of biotoxicity in water quality, and in some other embodiments it can also be referred to as a "water quality biotoxicity detection chip".

[0031] like Figure 1 As shown, the microfluidic chip may include: a chip body 10, microchannels, and micropore structures. Based on the microchannels and micropore structures, it can complete the entire process of water quality biotoxicity detection based on luminescent bacteria by orderly manipulating trace amounts of liquid.

[0032] The chip body 10 can be made of polydimethylsiloxane (PDMS) material through processing and modification. Specifically, it can be roughly cuboid in shape, formed by bonding two layers of PDMS. The area of ​​the upper surface is between 2 and 4 inches, and the thickness of each PDMS layer can be controlled to be about 3-5 mm. This chip design results in a smaller overall size, which reduces the overall throughput and thus the amount of bacterial solution required for each test.

[0033] Please continue reading. Figure 1 The chip body can be roughly divided into three regions: a mixing zone A1, a homogenization reaction zone A2, and a testing zone A3. Each region is equipped with corresponding microchannels and micropores to manipulate trace amounts of liquid and complete the detection process for water biotoxicity testing. The specific setup of each region is described below:

[0034] In the mixing region A1, the chip body is provided with: a first injection hole 101, a second injection hole 102, a first injection channel 103, a second injection channel 104, and a mixer 105.

[0035] The first injection port 101 can be connected to the mixer 105 via the first injection pipe 10320. The second injection port 102 can be connected to the mixer 105 via the second injection pipe 104. Thus, two independent injection channels are formed, which are used to inject the first liquid and the second liquid respectively, and the two are mixed in the mixer 105.

[0036] The first and second liquids are used only to distinguish liquids injected from different injection holes, and do not need to limit the specific composition of the liquids. The specific composition can be determined according to the method and procedures for water quality biotoxicity testing. For example, the first liquid can be a luminescent bacterial solution and a rinsing solution, and the second liquid can be a standard, a sample, and a rinsing solution. Preferably, in the mixing zone A1, the minimum channel width can be set to 5 μm, and the minimum channel height can be up to 10 μm.

[0037] In the mixing reaction zone A2, the chip body is provided with: reaction channel 201 and multiple reaction columns 202.

[0038] The reaction channel 201 is connected to the mixer 105, and the mixture in the mixer 105 (e.g., a mixture of luminescent bacteria and sample) can be further mixed and reacted in the reaction channel 201.

[0039] Specifically, the reaction channel 201 can be a serpentine channel with multiple bends, thereby providing sufficient channel length in a small area.

[0040] The reaction columns 202 are structures spaced apart along the extending direction of the reaction channel 201. They can cooperate with the reaction channel 201 to improve the mixing process. In some embodiments, such as Figure 2 As shown, the reaction column 202 can be configured as a cylindrical, triangular, or square column, etc. Different shapes and types of reaction columns can create different mixing and reaction effects, allowing technicians to design appropriate combinations according to actual needs.

[0041] Preferably, in the reaction channel 201, the channel spacing between adjacent reaction columns 202 can be controlled at 20 μm, and the minimum diameter of the reaction column can be controlled at 10 μm.

[0042] In this embodiment, the reaction column and reaction channel have dimensions at the micrometer scale, which can form a corridor reaction channel, thereby enabling efficient mixing. Furthermore, it can be combined with laminar and turbulent flow control to activate, clean, and mix processes in water quality biotoxicity detection.

[0043] Looping reaction refers to a process that uses circulating flow to achieve continuous processing and reaction of liquid samples. It is achieved through the design of specific channel structures, allowing the liquid sample to circulate within the channels, repeatedly passing through corresponding processing zones, thus achieving an efficient mixing and reaction process.

[0044] In actual testing, the mixture in mixer 105 (e.g., the mixture of sample and luminescent bacterial solution) can be continuously reacted and analyzed in multiple processing areas through different corridors or circulation paths in the reaction channel, thereby improving the throughput and efficiency of the experiment and reducing the waste of mixed liquid.

[0045] Please continue reading. Figure 1 In the test area A3, the chip body is equipped with: a detection ring 301, a drain hole 302, and a drain pipe 303.

[0046] The detection ring 301 is a cavity connected to the reaction channel 201. The mixed liquid, after being fully reacted and mixed in the reaction channel 201, can remain in the detection ring 301. The detection equipment can sense changes in the luminescence intensity within the detection ring 301 using a photoelectric detector, and obtain the results of the water quality biotoxicity test accordingly. The drain hole 302 is connected to the detection ring 301 via a drain pipe 303, allowing the liquid within the detection ring 301 to be discharged from the drain hole 302 after the test is completed.

[0047] In a preferred embodiment, such as Figure 3 As shown, in a chip body with a certain thickness, the detection ring 301 is closer to the upper surface of the chip body in the thickness direction compared to other microfluidic structures. This design allows emitted light to pass through the PDMS upper cover better and reach the corresponding photodetector.

[0048] Furthermore, the chip body 10 can also be covered with a black matte coating 11 in certain areas, thereby forming two parts in the chip body 10: a non-transparent area and a transparent area. The reaction channel can be arranged in the non-transparent area, while the detection ring 301 is located in the transparent area of ​​the chip body.

[0049] One advantage of this application embodiment is that by setting the above-mentioned black matte coating, inaccurate light in the mixing reaction area can be absorbed and blocked, thereby improving the accuracy of the detected luminescence intensity of the detection area.

[0050] To fully describe the inventive concept of this application, embodiments of this application also provide a method for preparing the above-mentioned water quality biotoxicity detection chip. Figure 4 This is a schematic diagram illustrating the preparation method provided in the embodiments of this application. Figure 4 As shown, the preparation process can be roughly divided into: preparing a chip mold (S410), pouring a liquid mixture into the chip mold and solidifying it (S420), separating and drilling holes in the solidified liquid mixture from the silicon wafer to obtain a PDMS layer with a channel structure on one surface (S430), bonding two PDMS layers to form a chip body with a closed channel structure (S440), and performing at least one material modification treatment on the chip body obtained after bonding (S450).

[0051] The following detailed explanation of each step of the above preparation method, using specific examples, is provided:

[0052] 1) Mold preparation:

[0053] First, the negative resist can be spin-coated onto a single-crystal silicon wafer. The spin-coating parameters can be set to 500 rpm for the front and 4000 rpm for the rear. After baking the silicon wafer at 65°C for 5 minutes, it is exposed to UV lithography using a Cr mask, then baked and spin-coated again for UV lithography (the mask must be aligned with the structure). After baking, it is soaked in developer for 10 minutes, and then thoroughly cleaned with a chromium removal solution.

[0054] Finally, the microstructure formed on the silicon wafer was hardened at 95°C for 10 minutes. The developer was a 0.5% NaOH aqueous solution. The chromium removal solution was prepared by dissolving cerium ammonium nitrate and 70% perchloric acid in distilled water.

[0055] 2) PDMS preparation and bonding:

[0056] The PDMS polymer was prepared by mixing monomers and curing agent AA3311 at a ratio of 10:1 and then stirring thoroughly. The resulting PDMS liquid mixture was then subjected to multiple degassing treatments in a vacuum dryer to remove surface air bubbles.

[0057] After the air bubbles disappear in the liquid mixture of PDMS and curing agent, the silicon wafer is wrapped in aluminum foil and then poured onto the silicon wafer with the microstructure formed. After pouring, it is placed on an oven set at 85°C and cured for 1 hour. After removal and cooling to room temperature, the PDMS layer is peeled off from the silicon wafer. By repeating the pouring and curing process twice using the same chip mold, the required two PDMS layers can be obtained.

[0058] Before performing microfluidic chip bonding, holes are first drilled in the PDMS layer at the locations where microporous structures (such as the first injection hole, the second injection hole, and the drain hole) need to be formed. Then, the side of the PDMS with the channel structure is cleaned. After cleaning, the PDMS can be placed in a plasma cleaner for 90 seconds to perform a hydrophilic treatment on the surface.

[0059] After hydrophilic treatment, the two PDMS layers are precisely bonded together to remove air bubbles inside the channel and form a closed channel structure. The bonded PDMS layers are then placed in an 80°C oven for 7 days to obtain the chip substrate.

[0060] 3) Material modification treatment (plasma modification treatment):

[0061] A custom-designed glass reactor with copper electrodes at the bottom, containing a 13.56MHz RF generator and impedance matching network, was used to place the fabricated chip body on top of the copper electrodes within the custom-designed glass reactor for plasma treatment.

[0062] The process involves establishing a base pressure of 1.5 Pa in a glass reactor using a pump, followed by the introduction of either argon (Ar) or nitrogen (N2) gas at a flow rate of 10 sccm, monitored by a vacuum gauge. The plasma power is 20 watts, and the treatment time ranges from 30 to 120 seconds. After plasma treatment, the chip body is cleaned with ultrapure water and dried with inert nitrogen.

[0063] It should be noted that this embodiment uses plasma modification as an example, but other material modification treatments can also be performed, and are not limited to plasma modification. For example, material modification can be achieved by increasing hydrophilic groups such as self-assembled peptides, hydroxyl groups, and epoxy groups to improve biocompatibility.

[0064] 4) Ultra-black matte coating processing:

[0065] In addition to material modification, a super-black matte coating can be applied to specific areas of the chip's surface. This coating is applied to the top, bottom, front, and back sides of these specific areas to create a non-transparent, covered region. For example, ... Figure 3 As shown, it can be coated on the upper and lower sides and the front and back sides of the mixing reaction area to form a non-transparent area, thus avoiding affecting the detection of the luminescence intensity of the detection ring.

[0066] In addition to improving the accuracy of detection, this ultra-black matte coating is also waterproof, resistant to high temperatures and corrosive substances, serving as an additional protective layer to ensure the lifespan of microfluidic chips and expand their application scenarios.

[0067] In the preparation method of this application embodiment, the PDMS material used possesses specific properties such as heat resistance, cold resistance, small viscosity change with temperature, water resistance, and non-toxicity, odorlessness, and breathability. Furthermore, PDMS material exhibits physiological inertness, good chemical stability, electrical insulation, and weather resistance. It has good hydrophobicity and high shear strength, allowing for long-term use at temperatures ranging from -50℃ to 200℃. It also has low surface tension and thermal conductivity, with a thermal conductivity coefficient of 0.134-0.159 W / (m·K). As a chip, it exhibits extremely high durability and has no negative impact on the overall reaction, nor does it have any biological inhibitory effects. In particular, PDMS material has 100% light transmittance and excellent optical transparency from 240 to 1100 nm, enabling highly sensitive detection characteristics without interfering with the PMT optical magnification detector.

[0068] PDMS materials can be modified to improve biocompatibility through methods such as plasma modification, self-contained peptide modification, or the addition of hydrophilic groups such as hydroxyl and epoxy groups. Increased biocompatibility allows for better adsorption and immobilization of bacterial strains, leading to more homogeneous mixing and reaction between the sample and the strain. This, in turn, regulates the overall detection process, resulting in more stable and accurate results.

[0069] Loctite AA3311 is a two-component system consisting of a base resin and a UV curing agent. When these two components are mixed and exposed to a UV light source, AA3311 rapidly cures, forming a strong bond. As a curing agent for PDMS chips, its physicochemical properties enhance the chip's biocompatibility, ensuring that the bacterial strain reacts with the sample without being inhibited by the chip.

[0070] Based on the aforementioned water quality biotoxicity detection chip, this application further provides a water quality biotoxicity detection system. It utilizes this water quality biotoxicity detection chip as the primary experimental step carrier to complete water quality biotoxicity detection based on luminescent bacteria. For example... Figure 5 As shown, the water quality biotoxicity detection system includes: a sample introduction module 510, a reagent storage module 520, a strain storage module 530, a reaction detection module 540, and a control module 550.

[0071] The sample introduction module 510 may include a primary homogenizer 511, a dilution and mixing chamber 512, and an ultrasonic homogenizer 513. It possesses methods for homogenization, fragmentation, dilution, and salinity adjustment, ensuring the integrity of the sample contents while avoiding clogging of the chip pathways and guaranteeing the accuracy of the results.

[0072] The sample introduction module 510 may also include a non-contact pump to achieve timed and quantitative sampling. The tubing in the sample introduction module 510 is selected using wear-resistant, corrosion-resistant, low-adsorption, and non-deformable connecting pipes to avoid the corrosion or adsorption of reagents and analytes that may affect the measurement results, thereby ensuring the accuracy of sample introduction for water samples, standard solutions, reagents, etc.

[0073] The reagent storage module 520 includes a pure water storage tank and a standard solution storage tank. In some embodiments, a saline storage tank may also be added as needed. Each storage tank may be equipped with an overflow port to prevent the stored liquid from overflowing.

[0074] The bacterial culture storage module 530 includes a bacterial culture reservoir with temperature regulation function. It can maintain the physiological activity of the bacterial culture through temperature control. The bacterial culture reservoir can also be equipped with condensate drainage and magnetic stirring functions, used to prevent condensate from accumulating and flowing into the bacterial culture reservoir and to prevent the bacterial culture from settling at the bottom of the reservoir through stirring, respectively.

[0075] The reaction detection module 540 includes a microfluidic chip and a photomultiplier tube (PMT) made of the aforementioned PDMS material. Both are housed within a temperature-controlled, light-shielding protective shield to ensure temperature stability during the reaction and prevent condensation from affecting the reaction detection. The internal components are corrosion-resistant and should be easy to install, replace, and clean. Furthermore, the PMT-based detector can output a stable detection signal and can achieve automated online analysis of the sample using a data acquisition card and data processing system.

[0076] The control module 550 includes a control center 551 and a data processing system 552. The control center 551 is the core of the entire system, possessing appropriate logical operation capabilities to perform basic control functions such as sample injection, reaction and discharge, cleaning, data processing, and display. Furthermore, the control center 551 can also record and provide feedback on abnormal information, automatically discharge analytes and reagents being tested before the power outage upon power restoration after an unexpected power failure, and automatically clean each channel and reset it to the restart testing state.

[0077] The data processing system 552 is used for processing test data. It can provide functions for data and operation log acquisition, storage, processing, display and output, as well as alarm functions for reagent balance, abnormal information, instrument failure, and test results exceeding the standard.

[0078] The following section provides a detailed description of the detection process of the aforementioned water quality biotoxicity detection system, using several specific examples.

[0079] Example 1: Water quality biotoxicity detection based on Vibrio qinghaiensis.

[0080] Sample testing process:

[0081] 1.1) Control the connecting valve to connect the inlet of the sample injection module of the water sample to be tested with its receiving port.

[0082] 1.2) Controlling the non-contact pump, the sample is first pumped into the primary homogenizer 511 to break up large particles. The homogenized sample is then pumped into the dilution and mixing tank 512. Subsequently, the sample is diluted with purified water from the purified water storage tank using the non-contact pump, and the salinity of the sample is adjusted by drawing brine from the brine storage tank (the osmotic pressure adjustment solution is an 80 g / L NaCl solution, and the NaCl solution concentration in the test system is 8 g / L). The dilution and adjustment parameter information is transmitted to the data processing system for integration and calculation. Finally, the non-contact pump pumps the treated sample from the dilution and mixing tank 512 into the ultrasonic fragmentation tank 513 for sample fragmentation.

[0083] 1.3) Connect the outlet of the sample injection module 510 to the receiving port of the reaction detection module using the control valve. Turn on the magnetic stirrer and continuously stir the bacterial solution to ensure uniform mixing. Recover the lyophilized Vibrio qinghaiensis powder according to the standard, with a recovery time of 5-30 minutes. Store the recovered Vibrio qinghaiensis bacterial solution in a container at 2-4℃. The shelf life of the luminescent bacterial solution is 7 days.

[0084] 1.4) Control the non-contact pump to draw pure water from the storage tank and inject it into the reference tank, and use the non-contact pump to draw brine from the storage tank and pump it into the reference tank to adjust the salinity.

[0085] 1.5) Control the bacterial suspension pathways of the sample channel and the reference channel to take equal amounts of bacterial suspension; start the motor and pull out the syringe to make the two syringes draw in a small amount of bacterial suspension respectively.

[0086] 1.6) Control the sample pathways of the sample channel and the reference channel to take equal amounts of the test water sample and the reference water sample, respectively. After the bacterial solution is sampled, the control center 551 switches the two multi-channel selection valves and starts the motor to continue pulling the syringes outward, so that the two syringes respectively draw in a quantitative amount of the test water sample or the reference water sample.

[0087] 1.7) Water samples obtained from the sample channel and reference channel are pushed to the sample chip and reference chip, respectively. The control center 551 can control the motor to drive the two syringes to maintain a certain flow rate.

[0088] 1.8) Turn on the photomultiplier tubes and measure the luminescence intensity of the mixture in the sample chip and the reference chip. After the drive motor pushes the sample in the syringe to the chip, the control center 551 turns on the first photomultiplier tube and the second photomultiplier tube to detect the luminescence intensity of the mixture in the sample chip and the reference chip respectively, and starts timing.

[0089] 1.9) When the injection volume reaches the chip capacity, the control center 551 controls the liquid injection to stop, and the signal acquisition module collects the detection signal output by the first photomultiplier tube and the second photomultiplier tube when the timer reaches time t, and converts it into corresponding light intensity data Iat and Ibt.

[0090] Where Iat represents the luminescence intensity of the mixture in the sample chip, and Ibt represents the luminescence intensity of the mixture in the reference chip. This luminescence intensity data can be used by the data acquisition card 553 and the data processing system 552 to complete the automatic online analysis of the sample.

[0091] Channel cleaning process:

[0092] 2.1) Control the injection channel and reference channel to draw sufficient pure water to clean the injection module.

[0093] 2.2) The pure water in the sample channel and the reference channel is pushed to the sample chip and the reference chip respectively to clean the reaction detection module.

[0094] 2.3) Discharge the cleaning waste liquid. Specifically, a water pump can be installed upstream of the waste liquid tank to extract the cleaning waste liquid from the sample chip and reference chip and collect it in the waste liquid tank.

[0095] Positive test process:

[0096] To ensure the parallelism of measurements, positive detection can be periodically initiated on the analyzer. Preferably, a positive detection can be performed every 10 samples, with the sample to be tested replaced with a standard solution pumped from the standard solution cell. If the relative luminescence inhibition rate (%) is less than -20%, the luminescent bacteria in the bacterial solution are considered normal and the parallelism of the analyzer meets the requirements; otherwise, the bacterial solution needs to be replaced or the analyzer needs to be checked for malfunction.

[0097] Example 2: Water biotoxicity detection based on the luminescent gene *Escherichia coli* lux:

[0098] Sample testing process:

[0099] 1.1) Control the connecting valve to connect the inlet of the sample injection module of the water sample to be tested with its receiving port.

[0100] 1.2) Controlling the non-contact pump, the sample is first pumped into the primary homogenizer 511 to break up large particles. Then, the homogenized sample is pumped into the dilution and mixing tank 512. Subsequently, the sample is diluted with pure water from the pure water storage tank using the non-contact pump. The dilution and adjustment parameter information is transmitted to the data processing system for integrated calculation. Finally, the non-contact pump pumps the processed sample from the dilution and mixing tank 512 into the ultrasonic fragmentation tank 513 for further fragmentation.

[0101] 1.3) Connect the outlet of the sample injection module 510 to the receiving port of the reaction detection module using the control valve. Turn on the magnetic stirrer and continuously stir the bacterial solution to ensure uniform mixing. Recover the lyophilized E. coli luminescent gene lux according to the standard, with a recovery time of 5-30 minutes. Store the recovered bacterial solution in a container at 2-4℃. The shelf life of the luminescent bacterial solution is 7-28 days.

[0102] 1.4) Control the non-contact pump to draw pure water from the storage tank and inject it into the reference tank.

[0103] 1.5) Control the bacterial suspension pathways of the sample channel and the reference channel to take equal amounts of bacterial suspension; start the motor and pull out the syringe to make the two syringes draw in a small amount of bacterial suspension respectively.

[0104] 1.6) Control the sample pathways of the sample channel and the reference channel to take equal amounts of the test water sample and the reference water sample, respectively. After the bacterial solution is sampled, the control center 551 switches the two multi-channel selection valves and starts the motor to continue pulling the syringes outward, so that the two syringes respectively draw in a quantitative amount of the test water sample or the reference water sample.

[0105] 1.7) Water samples obtained from the sample channel and reference channel are pushed to the sample chip and reference chip, respectively. The control center 551 can control the motor to drive the two syringes to maintain a certain flow rate.

[0106] 1.8) Turn on the photomultiplier tubes and measure the luminescence intensity of the mixture in the sample chip and the reference chip. After the drive motor pushes the sample in the syringe to the chip, the control center 551 turns on the first photomultiplier tube and the second photomultiplier tube to detect the luminescence intensity of the mixture in the sample chip and the reference chip respectively, and starts timing.

[0107] 1.9) When the injection volume reaches the chip capacity, the control center 551 controls the liquid injection to stop, and the signal acquisition module collects the detection signal output by the first photomultiplier tube and the second photomultiplier tube when the timer reaches time t, and converts it into corresponding light intensity data Iat and Ibt.

[0108] Where Iat represents the luminescence intensity of the mixture in the sample chip, and Ibt represents the luminescence intensity of the mixture in the reference chip. This luminescence intensity data can be used by the data acquisition card 553 and the data processing system 552 to complete the automatic online analysis of the sample.

[0109] Channel cleaning process:

[0110] 2.1) Control the injection channel and reference channel to draw sufficient pure water to clean the injection module.

[0111] 2.2) The pure water in the sample channel and the reference channel is pushed to the sample chip and the reference chip respectively to clean the reaction detection module.

[0112] 2.3) Discharge the cleaning waste liquid. Specifically, a water pump can be installed upstream of the waste liquid tank to extract the cleaning waste liquid from the sample chip and reference chip and collect it in the waste liquid tank.

[0113] Positive test process:

[0114] To ensure measurement parallelism, the analyzer needs to be periodically activated for positive detection, preferably once every 10 samples. The sample to be tested should be replaced with a standard solution pumped from the standard solution reservoir. If the relative luminescence rate (%) is around 50%, the luminescent bacteria in the bacterial solution are considered normal and the analyzer's parallelism meets the requirements; otherwise, the bacterial solution needs to be replaced or the analyzer needs to be checked for malfunction.

[0115] In summary, the embodiments of this application provide a microfluidic chip capable of mixing and reacting samples with bacterial solutions. Its overall surface area is controlled within the range of 2-4 inches, reducing the amount of bacterial solution required for each test. Furthermore, the PDMS-based microfluidic chip is easy to manufacture and inexpensive, and can be easily replaced. Therefore, when applied to a water quality biotoxicity detection system, it simplifies the replacement of the reaction module, facilitating regular replacement and ensuring detection accuracy.

[0116] Furthermore, this microfluidic chip utilizes hydrodynamic properties to form a corridor reaction channel in the reaction channel by designing a specific reaction column structure. Moreover, the overall throughput of the channel is very small, which can effectively shorten the reaction time during detection.

[0117] Furthermore, the corridor reaction channel can achieve rapid bacterial activation and high-efficiency mixing. It can also combine laminar and turbulent flow to regulate activation, cleaning, and mixing processes. Among them, cylindrical, triangular, and square prisms have different fluid dynamic characteristics, which can improve the mixing effect. Utilizing the different fluid dynamic structures of the three types of reaction columns can create different mixing and reaction effects.

[0118] Furthermore, by applying a black matte coating to absorb visible light and block inaccurate light from the mixing reaction zone, the PMT effectively captures the intensity changes of the detection zone, thereby improving detection accuracy. Moreover, this matte coating also serves as a protective layer, possessing waterproof, high-temperature resistance, and resistance to corrosive substances, ensuring service life and expanding application scenarios.

[0119] Furthermore, the PDMS material used to fabricate microfluidic chips possesses characteristics such as heat resistance, cold resistance, minimal viscosity variation with temperature, water resistance, low surface tension, and thermal conductivity (0.134-0.159 W / (m·K)). In particular, its excellent optical transparency (240-1100 nm) with 100% light transmittance provides highly sensitive detection characteristics without interfering with PMT (Polymerized Matrix Technology).

[0120] In addition, PDMS material is non-toxic, odorless, and breathable. It has physiological inertness, good chemical stability, electrical insulation and weather resistance, good hydrophobicity, and high shear strength. It can be used for a long time at temperatures ranging from -50℃ to 200℃. This makes the chips prepared with it extremely durable and has no negative effect on the overall reaction and no biological inhibition.

[0121] Furthermore, using UV-curable adhesive AA3311 as a curing agent for PDMS materials can improve the biocompatibility of the chip due to the material's physicochemical properties, ensuring that the bacterial strain reacts with the sample without being inhibited by the chip.

[0122] Furthermore, during the fabrication of the microfluidic chip, the PDMS material is modified using the principle of embedding and immobilization (including but not limited to: plasma modification; self-contained peptide modification; modification by adding hydrophilic groups such as hydroxyl and epoxy groups, etc.) to increase biocompatibility, which can adsorb and immobilize the strains and make the sample and strains mix and react better, resulting in more stable overall detection and more accurate detection results.

[0123] Furthermore, the water quality biotoxicity detection system incorporates a pretreatment process for samples. By methods such as stirring and crushing, the system ensures the integrity of the sample contents while avoiding clogging the channels inside the chip, thus guaranteeing the accuracy of the results.

[0124] Furthermore, this water quality biotoxicity detection system also has a control center, which can automate the operation and control of parameters such as sample mixing with bacterial solution and flow rate difference, reducing manpower and improving detection efficiency.

[0125] Furthermore, the water quality biotoxicity detection system is equipped with a dual-channel system consisting of two microfluidic chips, which ensures the continuity and timeliness of monitoring. Moreover, by setting up a dual-channel parallel detection method for samples and reference materials, the accuracy of the detection results can be improved.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them; under the concept of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of this application as described above, which are not provided in detail for the sake of brevity; although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A water quality biological toxicity detection chip, characterized in that, include: The chip body, the microchannels located inside the chip body, and the multiple micropore structures; The microporous structure includes: The first injection port is used to inject the first liquid; The second injection port is used to inject the second liquid; Drain hole, used to drain liquid; The microchannels include: The mixer is connected to the first injection port via a first injection conduit and to the second injection port via a second injection conduit; The detection ring is connected to the drain hole via a drain pipe; A reaction channel is located between the mixer and the detection coil; Several reaction columns; the several reaction columns are spaced apart along the extension direction of the reaction channel to form a corridor reaction channel; The reaction channel length between two adjacent reaction columns is 20 μm; the diameter of each reaction column is 10 μm.

2. The water quality biological toxicity detection chip according to claim 1, characterized in that, The reaction column is selected from one or more of the following: cylindrical, triangular, and square columns.

3. The water quality biological toxicity detection chip according to claim 1, characterized in that, The chip has a preset thickness; In this configuration, the detection ring, compared to other microfluidic structures, is closer to the surface of the chip body in the thickness direction.

4. The water quality biotoxicity detection chip according to claim 1, characterized in that, The minimum channel width of the first injection channel and the second injection channel is 5 μm; the minimum channel height of the first injection channel and the second injection channel is 10 μm.

5. The water quality biotoxicity detection chip according to claim 1, characterized in that, The chip body is formed by bonding two sheet-like structures; the sheet-like structures are made of PDMS material. The thickness of the sheet structure is 3-5 mm; the area of ​​the sheet structure is 2-4 inches.

6. The water quality biotoxicity detection chip according to claim 1, characterized in that, At least a portion of the chip body is covered with a black matte coating, forming a non-transparent area; The reaction channel is located within the non-transparent area; the detection ring is located within the transparent area of ​​the chip body.

7. A method for preparing a water quality biotoxicity detection chip as described in claim 1, characterized in that, include: Fabricating silicon wafer-based chip molds; A liquid mixture is poured into the chip mold; the liquid mixture is formed by uniformly mixing PDMS polymer monomers and curing agents in a preset ratio; The solidified liquid mixture is separated from the silicon wafer to obtain a PDMS layer, and microporous structures are formed by drilling holes at target locations. Two PDMS layers are bonded together to fabricate the chip body; The chip body undergoes at least one material modification treatment; the material modification treatment is selected from one or more of the following treatments: Plasma modification; or Add hydrophilic groups.

8. The preparation method according to claim 7, characterized in that, After performing at least one material modification treatment on the chip body, the method further includes: coating at least a portion of the surface of the chip body with a black coating to form a black matte coating; Before bonding the two PDMS layers, the method further includes: performing a hydrophilic treatment on the surface of the PDMS layers having microchannels.

9. A water quality biotoxicity detection system, characterized in that, include: The reaction detection module includes: a water quality biotoxicity detection chip as described in any one of claims 1-6 and a photomultiplier tube; The sample injection module is used to inject reagents into the water quality biotoxicity detection chip through the first injection port and the second injection port of the water quality biotoxicity detection chip; The reagent storage module is used to store reagents; A microbial storage module for storing bioluminescent bacteria; The control module is used to control the sample injection module to inject the sample and generate corresponding detection results based on the detection data of the photomultiplier tube.

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