A high-throughput evaluation system and method for bactericides and a microfluidic chip thereof

Abstract

The application discloses a high-throughput evaluation system and method for a bactericide and a micro-fluidic chip thereof. The micro-fluidic chip comprises two inlets, an adaptive damping type multi-stage concentration gradient generation module, a plurality of parallel test channels and a plurality of outlets in one-to-one correspondence with the plurality of test channels. The adaptive damping type multi-stage concentration gradient generation module comprises at least one level, each level comprises a vertical flow channel and a plurality of damping flow channels, and the plurality of damping flow channels are equidistantly distributed on the vertical flow channel and vertically communicated with the vertical flow channel. The two inlets are respectively communicated with the vertical flow channels in the adjacent levels. The damping flow channels in the last level are connected with the test channels through variable cross-section flow channels in one-to-one correspondence. The damping flow channels in each level are connected with the vertical flow channels of the adjacent next level through variable cross-section flow channels. Metal samples are embedded in each test channel. The application improves the high-throughput and precision of bactericide screening.

Description

Technical Field

[0001] This invention relates to the fields of microfluidics and microbial corrosion protection, and in particular to a high-throughput evaluation system and method for bactericides and its microfluidic chip. Background Technology

[0002] Microbial-induced corrosion (MIC) is considered a direct cause of severe economic losses in the oil and gas industry. Among corrosion-related microorganisms, sulfate-reducing bacteria (SRB) are considered the most prevalent and critical microorganisms initiating MIC. SRB are a type of anaerobic microorganism widely present in the natural environment. Especially in anaerobic environments such as oil pipelines, SRB reduce sulfate to sulfides through a series of complex biochemical reactions, thereby accelerating the corrosion of metallic materials. The presence of SRB not only alters the local environment around the pipeline but also promotes biofilm formation, further exacerbating the corrosion process. As a complex structure composed of SRB and other microorganisms, the biofilm adsorbs onto the pipeline surface, forming a local microenvironment that not only promotes the growth and reproduction of SRB but also significantly intensifies the corrosion reaction process.

[0003] Currently, pipeline corrosion protection technologies include bactericides, reinforcing agents, antibacterial stainless steel, and antibacterial coatings. Among these, using bactericides to eliminate corrosive microorganisms and inhibit their activity, thereby slowing down microbial corrosion on ferrous metals, is a common metal protection method. Traditional bactericide inhibition experiments mainly include the maximum probable number method (MPN method), broth dilution method (MIC / MBC determination), and the plate method. These methods involve cumbersome experimental operations and have limitations such as low throughput, low controllability, extremely long cycles (usually 7-21 days), and high costs. Furthermore, current equipment is bulky and cannot easily adapt to in-situ characterization techniques, while traditional biological characterization methods are time-consuming and material-intensive.

[0004] In response to the aforementioned challenges, the development of microfluidic technology has provided a revolutionary opportunity to understand the corrosion mechanism of SRB and develop novel prevention and control strategies. Microfluidic chip technology can integrate multiple operational units such as sample reaction, separation, and detection into a micro-device, offering numerous advantages such as fluid controllability, low reagent consumption, and rapid analysis and detection. In particular, the microenvironment within the chip can provide an excellent environment for bacterial cell growth, making it a valuable evaluation system for bacterial biofilm culture and research.

[0005] Although microfluidic technology has been introduced into microbial sensitivity testing and corrosion experiments, existing systems still face multiple limitations when dealing with the complex operating conditions of oilfields. Regarding high-throughput screening of agents, existing concentration gradient chips are mostly designed for aerobic or facultative anaerobic bacteria, lacking environmental adaptation for strict anaerobic bacteria (such as SRB). Meanwhile, detection devices based on electrochemistry or droplet splitting often rely on single-concentration sample introduction or focus on microscopic cell sorting, lacking the ability to generate multi-level gradients on-chip and simulate complex flow fields. In terms of reproducing interfacial corrosion behavior, some platforms use micro-wire probes, which cannot realistically replace the surface morphology and coating characteristics of solid carbon steel blocks in oilfields; other platforms, although integrating carbon steel working electrodes under laminar flow, have failed to achieve high-throughput parallel screening of bactericide concentration gradients. Furthermore, existing microfluidic systems rely heavily on low-integration external gas control or paraffin sealing systems to maintain anaerobic environments, and fluid control is mostly limited to low-shear force testing environments, making it difficult to realistically reproduce the harsh real-world conditions of strict anaerobic conditions coupled with high-velocity shear erosion within oil pipelines. Summary of the Invention

[0006] To address the technical problems existing in the background art, this invention proposes a high-throughput evaluation system and method for bactericides and its microfluidic chip.

[0007] In a first aspect, the present invention proposes a microfluidic chip, comprising two inlets, an adaptive damping multi-level concentration gradient generation module, multiple parallel test channels, and multiple outlets connected to the test channels one-to-one. The adaptive damping multi-level concentration gradient generation module adopts a multi-level branch topology, which includes at least one level. Each level includes a vertical flow channel and multiple damping flow channels, with the multiple damping flow channels equidistantly distributed on the vertical flow channel and perpendicularly connected to it. The two inlets are respectively connected to the vertical flow channels in their adjacent levels. The number of damping flow channels in the final level is the same as the number of test channels, and the damping flow channels in the final level are connected to the test channels one-to-one through variable cross-section flow channels. The damping flow channels in each level are connected to the vertical flow channels of the adjacent next level through variable cross-section flow channels. Each test channel contains a metal sample, and the top surface of the metal sample is coplanar with the bottom surface of the corresponding test channel.

[0008] Preferably, the inlet section of the test channel is provided with a local contraction structure for forming a high-shear flow field.

[0009] Preferably, each test channel is provided with an observation area, in which the metal sample is embedded.

[0010] Preferably, the microfluidic chip includes a cover plate and a base plate; a groove is formed on the bottom surface of the cover plate, and the cover plate is closed on the base plate to form a flow channel, with both the inlet and outlet on the cover plate; metal samples are embedded in the base plate at the corresponding positions of the corresponding test channels, and are seamlessly coplanar with the bottom surface of the test channels.

[0011] Preferably, the metal sample is made of N80 carbon steel.

[0012] In a second aspect, the present invention also proposes a high-throughput evaluation system for bactericides based on a microfluidic chip, comprising: an anaerobic closed environment control mechanism, a sample injection driving mechanism, a microfluidic chip as described in any one of the first aspects, a terminal multimodal characterization mechanism, and a data processing and evaluation mechanism. The sample injection drive mechanism is used to inject a preset bacterial solution and a preset bactericide into two inlets at the same flow rate, respectively. An adaptive damping multi-stage concentration gradient generator is used to create a bactericide concentration gradient across multiple test channels; Anaerobic closed environment control mechanism, used to provide the anaerobic environment required for sample introduction and static incubation; The terminal multimodal characterization mechanism includes a fluorescence detection mechanism and a morphology detection mechanism; the fluorescence detection mechanism performs fluorescence detection on each test channel after incubation to obtain the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel; the morphology detection mechanism is used to obtain the corrosion morphology of the metal samples in each test channel after incubation. The data processing and evaluation mechanism is used to couple and correlate the concentration gradient of bactericides, the distribution and spatial characteristics of live and dead bacteria in biofilms, and the corrosion morphology of metal samples in each test channel to construct an evaluation chain of bactericide antibacterial efficacy and metal corrosion. Based on the bactericide concentration in different test channels and the evaluation chain of bactericide antibacterial efficacy and metal corrosion, a correlation law between concentration gradient and corrosion degree is constructed. Based on the correlation law between concentration gradient and corrosion degree, the antibacterial and anticorrosive efficacy of the bactericide to be evaluated is evaluated.

[0013] Thirdly, this invention also proposes a high-throughput evaluation method for bactericides based on microfluidic chips, comprising: S1. Perform hydrophilic activation and sterilization pretreatment on the microfluidic chip as described in any one of the first aspects, and place it in an anaerobic closed environment. S2. Simultaneously inject bacterial solution and bactericide into the two inlets at the same flow rate to form a stable concentration gradient in each test channel. S3. After the sample is injected, the microfluidic chip is statically incubated. S4. After incubation, the microfluidic chip is removed from the anaerobic sealed environment. A fluorescence detection mechanism is used to perform fluorescence detection on each test channel of the incubated microfluidic chip to obtain the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel. A morphology detection machine is used to obtain the corrosion morphology of the metal samples of each test channel of the incubated microfluidic chip. S5. Couple the concentration gradient of bactericide, the distribution and spatial characteristics of live and dead bacteria in biofilm in each test channel with the corrosion morphology of metal samples to construct an evaluation chain of bactericide antibacterial efficacy-metal corrosion; construct the correlation law of concentration gradient-corrosion degree based on the bactericide concentration in different test channels and the evaluation chain of bactericide antibacterial efficacy-metal corrosion. S6. Based on the correlation between concentration gradient and corrosion degree, the antibacterial and antiseptic effects of the bactericide to be evaluated are assessed.

[0014] Preferably, during fluorescence detection, a non-destructive scanning method is performed using SYTO-9 / PI dual fluorescence staining combined with a confocal laser scanning microscope. Based on the fluorescence signal obtained from the scan, the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel are obtained.

[0015] Preferably, during morphology detection, the bacteria attached to the microfluidic chip are sequentially fixed, dehydrated in a gradient, and dried. After processing, the microfluidic chip is disassembled and subjected to scanning electron microscopy to obtain the corrosion morphology of each metal sample.

[0016] Preferably, during the injection process, two single-channel syringe pumps are used to simultaneously inject bacterial solution and bactericide into two inlets at the same flow rate.

[0017] Preferably, the flow rate is 6 mL / h.

[0018] The present invention provides a high-throughput evaluation system and method for bactericides, along with its microfluidic chip. The self-regulation of pressure fluctuations and flow rate changes within the microfluidic system is achieved through the long-path damping channel in the adaptive damping multi-stage concentration gradient generation module. Variable cross-section channels are introduced for connection between adjacent levels and between the final damping channel and the test channel. The flow rate transition and shear force reconstruction caused by the abrupt change in the width of the cross-section channel are used to adaptively adjust the pressure drop of each branch, so that multiple test channels can accurately form the preset bactericide concentration gradient. Furthermore, by integrating a concentration gradient generation mechanism and multiple parallel test channels with embedded metal samples into a microfluidic chip, along with a sample introduction drive mechanism, an anaerobic closed environment control mechanism, a terminal multimodal characterization mechanism, and a data processing and evaluation mechanism, and using preset bacterial solutions and preset bactericides as evaluation objects, this invention fully leverages the synergistic effect of component diffusion and mixing at the microscale and dynamic simulation of the macroscopic oil transportation environment. This results in the evaluation system achieving automatic generation of bactericide concentration gradients and a significantly shortened evaluation cycle (within 16 hours). This invention not only improves the high throughput and high precision of bactericide screening but also meets the practical needs of oil and gas field operations for rapid, low-volume, and visualized evaluation technologies. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of a microfluidic chip in one embodiment of the present invention.

[0020] Figure 2 This is a schematic diagram of the structure of a microfluidic chip in another embodiment of the present invention.

[0021] Figure 3 This is a schematic diagram of the fluid dynamics simulation of a microfluidic chip at a flow rate of 6 mL / h in one embodiment of the present invention; wherein, (a) represents a microfluidic chip with three channels, and (b) represents a microfluidic chip with five test channels.

[0022] Figure 4 The images show the metal sample of the microfluidic chip in one embodiment of the present invention before and after incubation in a bright field with added MIT bactericide for 16 h; where (a) is before incubation and (b) is after incubation.

[0023] Figure 5 The image shows a SEM image of a metal sample of a microfluidic chip in one embodiment of the present invention after incubation in MIT bactericide for 16 h; where (a) represents channel one; (b) represents channel two; and (c) represents channel three.

[0024] Figure 6 The diagram shows the distribution of live and dead bacteria in the biofilm of a microfluidic chip in one embodiment of the present invention after incubation in MIT bactericide for 16 h; where (a) indicates no bactericide, (b) indicates MIT bactericide, and the bactericide concentrations of channels one, two and three are 160, 100 and 40 ppm respectively. Figure 7 This is a spatial distribution diagram of live and dead bacteria in a microfluidic chip under pure bacterial solution conditions in one embodiment of the present invention; wherein, (a) represents channel one, (b) represents channel two, and (c) represents channel three.

[0025] Figure 8The spatial distribution diagram of live and dead bacteria of the microfluidic chip in one embodiment of the present invention after incubation in MIT bactericide for 16 h; wherein (a) represents channel one, (b) represents channel two, and (c) represents channel three.

[0026] Figure 9 The images show SEM images of metal samples in a pair of proportions proposed in this invention after incubation with MIT bactericide in a classic experimental platform for 16 h; where (a) represents channel one, (b) represents channel two, and (c) represents channel three. Detailed Implementation

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] In a first aspect, the present invention proposes a microfluidic chip, comprising two inlets 1, an adaptive damping multi-level concentration gradient generation module, multiple parallel test channels 3, and multiple outlets 4 connected to the multiple test channels 3 in a one-to-one correspondence; the adaptive damping multi-level concentration gradient generation module adopts a multi-level branch topology structure, which includes at least one level, each level including a vertical flow channel and multiple damping flow channels 2, and the multiple damping flow channels 2 are equidistantly distributed on the vertical flow channel and connected longitudinally to the vertical flow channel; the number of damping flow channels 2 in the final level is the same as the number of test channels 3, and the damping flow channels 2 in the final level are connected to the test channels 3 in a one-to-one correspondence through variable cross-section flow channels; the two inlets 1 are respectively connected to the vertical flow channels in the adjacent levels, and the damping flow channels 2 in each level are connected to the vertical flow channels of the next adjacent level through variable cross-section flow channels; each test channel 3 is embedded with a metal sample, and the top surface of the metal sample is coplanar with the bottom surface of the corresponding test channel 3.

[0029] It is important to understand that when fluid passes through the variable cross-section nodes of a multi-level network, the sudden contraction and expansion of the channel cross-section disrupts the original laminar velocity distribution, thereby generating corresponding local flow resistance. These local flow resistances, combined with the friction resistance of the long-path damped channel 2, form a flow resistance adaptive process, enabling the fluid to achieve uniform and constant flow distribution in each parallel test channel 3.

[0030] Furthermore, when fluid exits from the narrow damped flow channel 2 and enters the test channel 3 with a significantly increased cross-sectional area, according to the continuity equation... As the cross-sectional area A increases, the flow velocity V drops sharply. The extremely low and uniform flow velocity provides an ideal low-shear stress microenvironment for the surface of the embedded metal sample, which is highly conducive to the stable adhesion and uniform growth of the early SRB biofilm.

[0031] This invention achieves self-regulation of pressure fluctuations and flow rate changes within a microfluidic system through a long-path damping channel 2 in an adaptive damping multi-stage concentration gradient generation module. Variable cross-section channels are introduced between adjacent stages and between the final damping channel 2 and the test channel 3 for connection. Utilizing the flow rate transition and shear force reconstruction caused by abrupt changes in channel width, the pressure drop of each branch is adaptively adjusted, facilitating the precise formation of a preset bactericide concentration gradient across multiple test channels 3. This adaptive adjustment of the concentration gradient is achieved through a microchannel resistance distribution mechanism, eliminating random errors caused by manual preparation of agents and greatly benefiting the stable adhesion and uniform growth of SRB biofilms on metal samples in the early stages. Since the experiment is conducted only within microliter (μL) channels, the consumption of agents and expensive culture media is low. This micro-scale characteristic significantly reduces the screening cost of novel antibacterial materials and agents, resulting in significant economic benefits. Furthermore, within a flow rate range of 2-10 mL / h, the relative deviation of the concentration gradient is controlled within 5%.

[0032] It should be understood that in this embodiment, the top surface of the metal sample is coplanar with the bottom surface of the corresponding test channel 3, without steps or gaps, in order to avoid abnormal biofilm adhesion and flow field disturbance.

[0033] In some specific embodiments, the damping channel 2 is an S-shaped channel.

[0034] In some embodiments, the inlet section 1 of the test channel 3 is provided with a local contraction structure to form a high shear flow field.

[0035] In some specific embodiments, the microfluidic chip includes a cover plate and a base plate; a groove is formed on the bottom surface of the cover plate, and the cover plate is closed on the base plate to form a flow channel. Inlet 1 and outlet 4 are both formed on the cover plate; metal samples are embedded in the base plate at the corresponding positions of the corresponding test channels 3, and are seamlessly coplanar with the bottom surface of the test channels 3, without steps or gaps, so as to avoid abnormal biofilm adhesion and flow field disturbance.

[0036] Both the base plate and the cover plate are made of polydimethylsiloxane (PDMS) material. Specifically, a pre-prepared PDMS solution is poured onto a prepared SU-8 silicon wafer mold to create a cover plate containing flow channels. Simultaneously, a flow channel design diagram printed at a 1:1 scale is attached to the bottom of the base plate mold (culture dish) for reference positioning. N80 carbon steel is placed in the corresponding test channel 3, and molding continues to create a base plate containing N80. After molding, both are baked at 60 ℃ for 3 h. After complete curing, they are peeled from the culture dish and cut to a size suitable for subsequent assembly. Holes are punched in the cover plate with flow channels. Both are placed in a plasma cleaner for plasma treatment (with the surfaces to be bonded facing upwards), removed, and bonded. Finally, sterile deionized water is injected into the microfluidic chip to test its airtightness.

[0037] In some specific embodiments, the channel has a height of 100 μm and a width of 200-400 μm.

[0038] In some specific embodiments, the metal sample is made of N80 carbon steel.

[0039] like Figure 1 As shown, in some specific embodiments, the concentration gradient generating mechanism is a three-stage concentration gradient generator, which can form a three-stage concentration band that decreases along the path, and can generate a gradient difference of approximately 30% of the fungicide concentration distribution in multiple test channels 3.

[0040] like Figure 2 As shown, in some specific embodiments, the concentration gradient generating mechanism is a five-level concentration gradient generator, which can form a five-level concentration band that decreases along the path, and can generate a bactericide concentration distribution with a gradient difference of about 20% in multiple test channels 3.

[0041] In some specific embodiments, for Figure 1 and Figure 2 The microfluidic chip in the study examined the coupling relationship between flow rate and concentration. Two mass concentrations were set for inlet 1 (200 ppm and 100 ppm), while inlet 12 was pure solvent (0 ppm). The total flow rate was set to 6 mL / h, and the steady-state concentration at outlet 4 was recorded. Figure 3 It can be seen that, under the premise of uniform velocity, the two structures achieved concentration gradients of 0.30 and 0.20 in the experimental group, providing a good hydrodynamic basis for subsequent precise dose control.

[0042] In some embodiments, each test channel 3 is provided with an observation area, in which a metal sample is embedded.

[0043] This embodiment allows for real-time bright-field observation through the observation area, enabling real-time monitoring of the experimental process and providing a highly standardized and dynamically controllable experimental platform for high-throughput evaluation of fungicides.

[0044] Secondly, referring to Figure 1 The present invention also proposes a high-throughput evaluation system for bactericides based on microfluidic chips, comprising: an anaerobic closed environment control mechanism, a sample injection driving mechanism, a microfluidic chip as described in any one of the first aspects, a terminal multimodal characterization mechanism, and a data processing and evaluation mechanism; The sample injection drive mechanism is used to inject a preset bacterial solution and a preset bactericide into the two inlets 1 at the same flow rate; An adaptive damping multi-stage concentration gradient generator is used to create a bactericide concentration gradient across multiple test channels 3. An anaerobic closed-loop environment control system is used to provide the anaerobic environment required for sample introduction and static incubation. The terminal multimodal characterization mechanism includes a fluorescence detection mechanism and a morphology detection mechanism; the fluorescence detection mechanism performs fluorescence detection on each test channel 3 after incubation to obtain the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel 3; the morphology detection mechanism is used to obtain the corrosion morphology of the metal samples in each test channel 3 after incubation. The data processing and evaluation mechanism is used to couple and correlate the concentration gradient of bactericides, the distribution and spatial characteristics of live and dead bacteria in biofilms, and the corrosion morphology of metal samples in each test channel 3 to construct an evaluation chain of bactericide antibacterial efficacy-metal corrosion. Based on the bactericide concentration in different test channels 3 and the evaluation chain of bactericide antibacterial efficacy-metal corrosion, a correlation law of concentration gradient-corrosion degree is constructed. Based on the correlation law of concentration gradient-corrosion degree, the antibacterial and anticorrosive efficacy of the bactericide to be evaluated is evaluated.

[0045] This invention establishes a functional system integrating fluid drive, environmental control, and open-field in-situ observation and characterization through an anaerobic closed environment control mechanism, a sample introduction drive mechanism, a microfluidic chip, a terminal multimodal characterization mechanism, and a data processing and evaluation mechanism. The sample introduction drive mechanism enables precise control of the flow rate of the preset bacterial solution and the preset bactericide. The anaerobic closed environment control mechanism creates the growth environment required for bacterial growth in the preset bacterial solution. The microfluidic chip dynamically stabilizes the incubation temperature in an anaerobic environment that meets the temperature and environmental parameters required for generation. This provides a highly standardized and dynamically controllable experimental platform for high-throughput evaluation of the bactericide. The terminal multimodal characterization mechanism obtains the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel 3, as well as the corrosion morphology of the metal samples. The data processing and evaluation mechanism evaluates the antibacterial and antiseptic efficacy of the bactericide based on the characterization results of each test channel 3.

[0046] It should be noted that the bacteria in the bacterial solution in this embodiment are strictly anaerobic bacteria.

[0047] In some specific embodiments, for oilfield pipelines, the bacteria are pre-selected as sulfate-reducing bacteria (SRB).

[0048] For oilfield pipelines, this invention leverages the micro-space effect of microfluidic chips to significantly increase the specific surface area, inducing sulfate-reducing bacteria (SRB) to rapidly complete the entire process from initial attachment to mature biofilm evolution at the metal interface. Through microscale acceleration, the evaluation cycle of traditional methods (such as tablet and bottle testing) is successfully shortened from 14-28 days to less than 16 hours. This breakthrough not only provides rapid decision support for agent replacement in oilfields but also demonstrates the technical feasibility for large-scale, high-throughput initial screening of bactericides.

[0049] This invention not only precisely controls the linear diffusion and mixing of bactericides using a microfluidic chip, but also accurately recreates the real flow field shear force environment of the oil pipeline wall through flow rate regulation of the injection mechanism. This system coupling effect of precise microscale diffusion and macroscopic industrial condition simulation effectively solves the problem of experimental data distortion caused by the lack of fluid dynamic interference in traditional static experiments, ensuring a high degree of consistency and extrapolability between laboratory evaluation results and field conditions in oil and gas fields.

[0050] In some embodiments, the anaerobic closed environment control unit is an oxygen-free constant temperature chamber filled with high-purity nitrogen gas and the temperature is controlled at 37°C, which is suitable for the strictly anaerobic incubation environment preset in the experiment.

[0051] Thirdly, this invention also proposes a high-throughput evaluation method for bactericides based on microfluidic chips, comprising: S1. Perform hydrophilic activation and sterilization pretreatment on the microfluidic chip as described in any one of the first aspects, and place it in an anaerobic closed environment. S2. Simultaneously inject bacterial solution and bactericide into the two inlets 1 at the same flow rate to form a stable concentration gradient in each test channel 3. S3. After the sample is injected, the microfluidic chip is statically incubated. S4. After incubation, the microfluidic chip is removed from the anaerobic sealed environment. A fluorescence detection mechanism is used to perform fluorescence detection on each test channel 3 of the incubated microfluidic chip to obtain the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel 3. A morphology detection machine is used to obtain the corrosion morphology of the metal samples of each test channel 3 of the incubated microfluidic chip. S5. Couple the concentration gradient of bactericide, distribution and spatial characteristics of live and dead bacteria in biofilm in each test channel 3 with the corrosion morphology of metal samples to construct an evaluation chain of bactericide antibacterial efficacy-metal corrosion; construct the correlation law of concentration gradient-corrosion degree based on the bactericide concentration in different test channels 3 and the evaluation chain of bactericide antibacterial efficacy-metal corrosion. S6. Based on the correlation between concentration gradient and corrosion degree, the antibacterial and antiseptic effects of the bactericide to be evaluated are assessed.

[0052] In the fluorescence detection, the SYTO-9 / PI dual fluorescence staining method was combined with a confocal laser scanning microscope for non-destructive scanning. Based on the fluorescence signal obtained from the scan, the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel 3 were obtained.

[0053] In the morphology detection process, the bacteria attached to the microfluidic chip were sequentially fixed, dehydrated in a gradient, and dried. After the treatment was completed, the microfluidic chip was disassembled and subjected to scanning electron microscopy to obtain the corrosion morphology of each metal sample.

[0054] During the injection process, two single-channel injection pumps are used to simultaneously inject bacterial solution and bactericide into the two inlets 1 at the same flow rate, so as to form a stable concentration gradient in each test channel 3.

[0055] In some embodiments, the flow rate is 2-10 mL / h.

[0056] In some specific embodiments, the flow rate is 6 mL / h.

[0057] The present invention will now be described in conjunction with specific embodiments.

[0058] Example 1

[0059] like Figure 1 As shown, this embodiment discloses a high-throughput evaluation method for bactericides based on microfluidic chips, including: S1. The microfluidic chip undergoes hydrophilic activation and sterilization pretreatment, and is then placed in an anaerobic, sealed environment. The microfluidic chip includes a base plate and a cover plate. A groove is formed on the bottom surface of the cover plate, which closes onto the base plate to form a flow channel. The flow channel includes two inlets 1, an adaptive damping multi-level concentration gradient generation module, multiple parallel test channels 3, and multiple outlets 4 connected to each test channel 3. The adaptive damping multi-level concentration gradient generation module adopts a multi-level branch topology, which includes at least one level. Each level includes a vertical flow channel and multiple damping flow channels 2, with the multiple damping flow channels 2 equidistantly distributed on the vertical flow channel and longitudinally connected to it. The number of damping flow channels 2 in the final level is the same as the number of test channels 3, and the final level... The damping flow channel 2 and the test channel 3 are connected one-to-one through a variable cross-section flow channel; the two inlets 1 are connected to the vertical flow channels in the adjacent layers respectively, and the damping flow channel 2 in each layer is connected to the vertical flow channel of the next adjacent layer through a variable cross-section flow channel; each test channel 3 is provided with an observation area; the inlet 11 and outlet 4 are both opened on the cover plate, and the bottom plate and the corresponding position of each observation area are embedded with metal samples made of N80 carbon steel, and the top surface of the metal sample is coplanar with the bottom surface of the corresponding observation area; the adaptive damping multi-level concentration gradient generation module 2 is used to generate a linearly distributed bactericide concentration gradient in multiple test channels 3; among them, the adaptive damping multi-level concentration gradient generation module 2 is a three-level concentration gradient generator, and the test channels 3 are channel one, channel two and channel three from top to bottom; S2. Connect the inlet 1 of the microfluidic chip to the syringes on the two single-channel injection pumps respectively, and connect its outlet 4 to the discharge tubing; use the two single-channel injection pumps to simultaneously inject bacterial solution and bactericide into the two inlets 1 at a rate of 6 mL / h, so as to form a stable concentration gradient in each test channel 3. S3. When liquid flows out of the conduit connected to the outlet 4 of the microfluidic chip, stop the injection; after the injection is completed, perform static incubation of the microfluidic chip at 37 °C for 16 hours. S4. After incubation, the microfluidic chip was removed from the anaerobic sealed environment and non-destructively scanned using SYTO-9 / PI dual fluorescence staining combined with confocal laser scanning microscopy. Based on the fluorescence signal obtained from the scan, the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel 3 were obtained. The bacteria attached to the microfluidic chip were then fixed, dehydrated in a gradient, and dried in sequence. After processing, the microfluidic chip was disassembled and tested by scanning electron microscopy to obtain the corrosion morphology of each metal sample. S5. Couple the concentration gradient of bactericide, distribution and spatial characteristics of live and dead bacteria in biofilm in each test channel 3 with the corrosion morphology of metal samples to construct an evaluation chain of bactericide antibacterial efficacy-metal corrosion; construct the correlation law of concentration gradient-corrosion degree based on the bactericide concentration in different test channels 3 and the evaluation chain of bactericide antibacterial efficacy-metal corrosion. S6. Based on the correlation between concentration gradient and corrosion degree, the antibacterial and antiseptic effects of the bactericide to be evaluated are assessed.

[0060] from Figure 4 and Figure 5 It can be seen that corrosion is slight in the upstream region with higher bactericide concentrations, while corrosion is significantly aggravated in the downstream region with lower concentrations, with corrosion products developing from sparse particles to dense deposits and even localized pits; this is consistent with the corrosion observed in the second morphology along the concentration gradient in the flow channel under a bright-field microscope. Meanwhile, in... Figure 7 and Figure 8 The confocal fluorescence analysis shown indicates that the bacterial density is higher in the bottom and top regions of test channel 3, and lower in the middle region, exhibiting a V-shaped three-dimensional distribution. Furthermore, the biofilm shows a clear distribution of live and dead bacteria; the proportion of live bacteria increases as the bactericide concentration decreases. This is highly consistent with the corrosion levels observed by SEM, confirming that microbial activity is a key factor driving corrosion. Increased bactericide concentration leads to a decrease in total bacterial density, thus significantly mitigating corrosion damage on the N80 carbon steel surface. In addition, the results of this study are compared with those of classic traditional platforms (such as...). Figure 9 A comparison of experiments (incubated for 16 hours) revealed that the traditional platform also exhibited a corrosion inhibition effect due to the coverage of agglomerated corrosion products in the corresponding high-concentration group, while a large amount of SRB enrichment and pitted corrosion appeared in the low-concentration group. This result fully confirms that the evaluation results of the microfluidic system are highly consistent and reliable with those of macroscopic traditional experiments.

[0061] Example 2

[0062] This embodiment uses a microfluidic chip-based high-throughput evaluation method for bactericides to rapidly evaluate a single bactericide, MIT (isothiazolinone). The high-throughput evaluation system for bactericides is the same as in Embodiment 1, except that the preset initial concentration of the bactericide is 200 ppm.

[0063] Example 3

[0064] This embodiment uses a microfluidic chip-based high-throughput evaluation method for bactericides to evaluate the high throughput of a single bactericide, THPS (tetramethylhydroxyphosphine sulfate). The bactericide high-throughput evaluation system used is the same as in Example 1, except that the initial concentration of the bactericide is 200 ppm.

[0065] Example 4

[0066] This embodiment uses a microfluidic chip-based high-throughput evaluation method for bactericides to perform high-throughput evaluation of a single bactericide, PHMG (polyhexamethylene guanidine hydrochloride). The bactericide high-throughput evaluation system used is the same as in Example 1, except that the initial concentration of the bactericide is 200 ppm.

[0067] Example 5

[0068] This embodiment uses a microfluidic chip-based high-throughput evaluation method for bactericides to evaluate the high throughput of a single bactericide, SW-80. The system and method used are the same as in Example 1, except that the initial concentration of the bactericide is 200 ppm.

[0069] Example 6

[0070] A high-throughput evaluation method for bactericides based on microfluidic chips was used to evaluate the high-throughput of a compound bactericide (SJ-66+SJ-99) formed by dodecyl dimethyl benzyl ammonium chloride and glutaraldehyde (SJ-66+SJ-99). The high-throughput evaluation system for bactericides used was the same as in Example 1, except that the initial concentration of the bactericide was 100 ppm.

[0071] Comparative Example 1 Based on the evaluation of the single bactericide MIT (isothiazolinone) using traditional platforms, bacterial suspension under the same conditions as described in Example 1 and 100 ppm of bactericide solution were injected into anaerobic vials. After static incubation for 16 hours, the test pieces were removed, subjected to bacterial fixation, dehydration, and drying, and their surface morphology was observed using SEM. The SEM results were consistent with those obtained after 16 hours of incubation in a microfluidic system.

[0072] Comparative Example 2 Based on the evaluation of the compound bactericide (SJ-66+SJ-99) using a traditional platform, the bacterial suspension described in Example 1 and a solution of the two bactericides mixed at 100 ppm were injected into anaerobic vials. After static incubation for 16 h, the test pieces were removed, subjected to bacterial fixation, dehydration, and drying, and their surface morphology was observed using SEM. The SEM results were consistent with those obtained after 16 h of incubation in a microfluidic system.

[0073] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A microfluidic chip, characterized in that, It includes two inlets, an adaptive damped multi-level concentration gradient generation module, multiple parallel test channels, and multiple outlets that are connected to the test channels one by one. The adaptive damping multi-level concentration gradient generation module adopts a multi-level branching topology, which includes at least one level. Each level includes a vertical flow channel and multiple damping flow channels, with the multiple damping flow channels equidistantly distributed on the vertical flow channel and vertically connected to it. Two inlets are connected to the vertical flow channels in their adjacent levels. The number of damping flow channels in the final level is the same as the number of test channels, and the damping flow channels in the final level are connected to the test channels one-to-one through variable cross-section flow channels. The damping flow channels in each level are connected to the vertical flow channels of the next adjacent level through variable cross-section flow channels. Each test channel contains a metal sample, and the top surface of the metal sample is coplanar with the bottom surface of the corresponding test channel.

2. The microfluidic chip according to claim 1, characterized in that, The inlet section of the test channel is equipped with a localized contraction structure to create a high-shear flow field.

3. The microfluidic chip according to claim 1, characterized in that, The microfluidic chip includes a cover plate and a base plate; a groove is formed on the bottom surface of the cover plate, and the cover plate is closed on the base plate to form a flow channel. The inlet and outlet are both located on the cover plate; metal samples are embedded in the base plate at the corresponding positions of the corresponding test channels, and are seamlessly coplanar with the bottom surface of the test channels.

4. The microfluidic chip according to claim 1, characterized in that, The metal sample was made of N80 carbon steel.

5. The microfluidic chip according to claim 1, characterized in that, Each test channel has an observation area where the metal sample is embedded.

6. A high-throughput evaluation system for bactericides based on microfluidic chips, characterized in that, include: Anaerobic closed environment control mechanism, sample introduction drive mechanism, microfluidic chip as described in any one of claims 1-5, terminal multimodal characterization mechanism and data processing and evaluation mechanism; The sample injection drive mechanism is used to inject a preset bacterial solution and a preset bactericide into two inlets at the same flow rate, respectively. An adaptive damping multi-level concentration gradient generation module is used to generate a bactericide concentration gradient across multiple test channels; Anaerobic closed environment control mechanism, used to provide the anaerobic environment required for sample introduction and static incubation; The terminal multimodal characterization mechanism includes a fluorescence detection mechanism and a morphology detection mechanism; the fluorescence detection mechanism performs fluorescence detection on each test channel after incubation to obtain the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel; The morphology inspection unit is used to obtain the corrosion morphology of metal samples in each test channel after incubation. The data processing and evaluation mechanism is used to couple and correlate the concentration gradient of bactericides, the distribution and spatial characteristics of live and dead bacteria in biofilms, and the corrosion morphology of metal samples in each test channel to construct an evaluation chain of bactericide antibacterial efficacy and metal corrosion. Based on the bactericide concentration in different test channels and the evaluation chain of bactericide antibacterial efficacy and metal corrosion, a correlation law between concentration gradient and corrosion degree is constructed. Based on the correlation law between concentration gradient and corrosion degree, the antibacterial and anticorrosive efficacy of the bactericide to be evaluated is evaluated.

7. A high-throughput evaluation method for bactericides based on microfluidic chips, characterized in that, include: S1. Perform hydrophilic activation and sterilization pretreatment on the microfluidic chip as described in any one of claims 1-5, and place it in an anaerobic closed environment; S2. Simultaneously inject bacterial solution and bactericide into the two inlets at the same flow rate to form a stable concentration gradient in each test channel. S3. After the sample is injected, the microfluidic chip is statically incubated. S4. After incubation, the microfluidic chip is removed from the anaerobic sealed environment. A fluorescence detection mechanism is used to perform fluorescence detection on each test channel of the incubated microfluidic chip to obtain the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel. A morphology detection machine is used to obtain the corrosion morphology of the metal samples of each test channel of the incubated microfluidic chip. S5. Couple the concentration gradient of bactericide, the distribution and spatial characteristics of live and dead bacteria in biofilm in each test channel with the corrosion morphology of metal samples to construct an evaluation chain of bactericide antibacterial efficacy-metal corrosion; construct the correlation law of concentration gradient-corrosion degree based on the bactericide concentration in different test channels and the evaluation chain of bactericide antibacterial efficacy-metal corrosion. S6. Based on the correlation between concentration gradient and corrosion degree, the antibacterial and antiseptic effects of the bactericide to be evaluated are assessed.

8. The high-throughput evaluation method for bactericides based on microfluidic chips according to claim 7, characterized in that, During fluorescence detection, a non-destructive scanning method was performed using SYTO-9 / PI dual fluorescence staining combined with confocal laser scanning microscopy. Based on the fluorescence signal obtained from the scan, the distribution and spatial characteristics of live and dead bacteria in the biofilm of each test channel were obtained. During morphology detection, the bacteria attached to the microfluidic chip were fixed, dehydrated in a gradient, and dried in sequence. After the treatment, the microfluidic chip was disassembled and subjected to scanning electron microscopy to obtain the corrosion morphology of each metal sample.

9. The high-throughput evaluation method for bactericides based on microfluidic chips according to claim 7, characterized in that, During the injection process, two single-channel syringe pumps are used to simultaneously inject bacterial solution and bactericide into two inlets at the same flow rate; the flow rate is 2-10 mL / h.

10. The high-throughput evaluation method for bactericides based on microfluidic chips according to claim 7, characterized in that, The flow rate is 6 mL / h.

Images