A magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with ultra-slippery surface and use method

Abstract

The present invention provides a magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-smooth surface and a method of use, which belongs to the field of microbial detection technology. The microfluidic chip detection platform described in the present invention is an application of the integration of three research direction technologies: super-hydrophobic anti-biological adhesion materials, magnetically controlled separation and sorting, and electrochemical microbial Coulter counting detection sensors. The super-hydrophobic anti-biological adhesion material can increase liquid flux and avoid sample residue. The present invention combines immune nano-magnetic beads with magnetically controlled sorting functions to improve sorting efficiency; the present invention adopts a labeled fish pathogenic microorganism Coulter counting technology, and uses a Coulter counting method that generates instantaneous fluctuation pulses of current due to conductivity changes to achieve quantitative detection of fish pathogenic microorganisms. Compared with traditional laboratory incubation methods, microscope counting methods, etc., it has the advantages of less sample consumption and shorter time consumption.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganism detection, and in particular to a magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface and a use method thereof. Background Art

[0002] In response to bacterial diseases in marine aquaculture, researchers from various countries have been working extensively to develop rapid, accurate and effective diagnostic methods. Currently, the main methods include microbial physiological and biochemical detection, molecular biology methods, immunological methods and biochips.

[0003] Among them, the microbial physiological and biochemical detection methods are mainly based on morphological detection, physiology and a series of physiological and biochemical experiments to achieve preliminary identification of microorganisms. Its advantages are: accurate detection, ability to make comprehensive judgments through multiple indicators, more flexible, and more widely practiced; its disadvantages are: time-consuming, cumbersome operation, and not conducive to the rapid diagnosis of bacterial diseases. Molecular biological methods mainly include: polymerase chain reaction (PCR) technology, nucleic acid hybridization technology, etc. Its advantages are: high detection sensitivity, which is the technology with the highest detection sensitivity at present; its disadvantages are: expensive equipment, collected samples need to be taken back to the laboratory for culture and identification, and professional technicians are required to prepare samples and analyze results; at the same time, molecular detection technology cannot accurately judge the activity of microorganisms in samples. Immunological methods mainly include enzyme-linked immunosorbent assay (ELISA), immunochromatography technology, etc. Its advantages are: strong specificity, high sensitivity, and relatively stable detection effect; its disadvantages are: the immune operation process is time-consuming and cumbersome, and it is impossible to achieve real-time detection of samples. The biochip method mainly includes gene chips and protein chips, etc. Its advantages are: high-throughput parallel detection, small errors in human operation, high specificity, and high throughput; its disadvantages are: expensive, high cost, difficult protein synthesis and extremely easy to denature and inactivate, which in turn affects its properties and functions.

[0004] Moreover, at present, the detection of pathogenic microorganisms in marine ranches at home and abroad mainly focuses on the analysis and detection of tissues or organs of marine ranch breeding objects, while the high-throughput rapid detection technology and engineering intelligent equipment for pathogenic microorganisms in marine ranch environments are relatively lacking, which cannot meet the actual requirements of marine ranch biological disease prevention and control. Therefore, in view of the current situation that pathogenic microorganisms in marine ranch-farmed fish have a rapid onset and high mortality rate, there is an urgent need for a rapid, accurate, highly sensitive, and rapid identification of marine ranch microbial pathogens. Summary of the invention

[0005] In view of this, the present invention provides a magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface and a method of use. The detection platform is an integrated application of three research direction technologies: super-hydrophobic anti-biological adhesion material, magnetically controlled separation and sorting, and electrochemical microbial Coulter counting detection sensor.

[0006] The present invention couples the prepared super-slip coating material with the surface of a PDMS microfluidic chip doped with hydrophobic nano-silicon dioxide particles, so that a self-assembled lubricating layer is generated on the inner surface of the microfluidic chip channel. 2 The chemical reaction between the surface hydroxyl groups and the superlubricating material forms a bristle layer-like long-chain structure, which realizes the sliding transport of liquid samples in the channel.

[0007] The common sample residue clogging problem at 90°, 120°, and 180° positions of the traditional PDMS material microfluidic chip channel is solved by the low resistance of the inner surface super-slippery coating and repeated flushing of the cleaning solution. Since the chip channel surface preparation method provided by the present invention has the super-slippery property of the liquid phase working fluid, the droplets and the samples contained in the droplets cannot be retained at the corners of the channel. This function is particularly effective in the mixed reaction area and the magnetic control sorting area, and the durability of the super-slippery coating structure of the present invention is good.

[0008] Combined with the qualitative magnetic control separation of pathogenic microorganism-specific antibodies coupled to magnetic beads in the "Y"-shaped sorting section of the microfluidic chip, the subsequent enrichment of droplets containing fish pathogenic microorganisms is achieved, and the quantitative detection of fish pathogenic microorganisms is achieved through the Coulter counting at the end of the chip detection platform of the present invention, providing key technical support for the manufacture of high-throughput fish pathogenic microorganism microfluidic detection equipment.

[0009] A magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface, the detection platform comprising a PDMS microfluidic chip body.

[0010] The PDMS microfluidic chip body comprises: an organic buffer inlet (1), a sample stock solution inlet (2), a magnetic bead antibody mixed solution inlet (3), a mixed reaction area (4), a qualitative magnetic control sorting area (5), an enrichment area (6), a quantitative counting area (7) and a waste liquid outlet (8).

[0011] Furthermore, the mixed reaction zone (4) is PDMS@SiO 2 Substrate self-assembled long-chain superlubricating microchannels.

[0012] The PDMS@SiO 2 The preparation method of the substrate self-assembled long-chain superlubricating microchannel comprises:

[0013] S1, SiO 2The nanoparticles were added to toluene and stirred evenly to obtain SiO 2 A mixed solution in which nanoparticles are fully dispersed in toluene;

[0014] S2, after the PDMS and the mixed solution are stirred evenly, toluene is removed, and then the PDMS and the curing agent are mixed in a mass ratio of 7:1-9:1 to obtain a mixture, and then the mixture is degassed, and then the degassed mixture is poured into a mold to form a PDMS@SiO 2 substrate;

[0015] S3, PDMS@SiO 2 The substrate was immersed in an alkaline ethanol solution with a pH of ≥ 8.5, and then the PDMS@SiO 2 The substrate is air-dried, and then the air-dried substrate is modified using plasma;

[0016] S4. After modification, PDMS@SiO 2 The substrate surface was sprayed with a mixture of toluene and DCDMS for hydrolysis reaction, and then repeatedly rinsed with toluene solution and dried at room temperature to obtain PDMS@SiO 2 Substrate self-assembled long-chain superlubricating microchannels.

[0017] Furthermore, the SiO 2 The mass ratio of nanoparticles, PDMS and toluene is 5:8:87.2-8:10:87.2.

[0018] Furthermore, in step S2, the temperature of the mold is 85-100° C., and the heating is performed for 1-1.5 hours.

[0019] Furthermore, the immersion time in step S3 is 45-60 min, the modification time in step S3 is 5-10 min, and the O 2 The concentration is 65%.

[0020] Furthermore, the hydrolysis reaction time in step S4 is 45-60 min.

[0021] Furthermore, the qualitative magnetic control sorting area includes capturing pathogenic microorganisms using immune nanomagnetic beads.

[0022] Furthermore, the quantitative counting area uses a Coulter counting method that uses conductivity changes to generate instantaneous fluctuation pulses of current to achieve quantitative analysis and detection of pathogenic microorganisms in the sample.

[0023] Furthermore, the channel width from the organic buffer inlet to the mixed reaction zone is 50±5 μm, the channel width of the qualitative magnetic control separation zone is 100±10 μm, and the length is 1.5-2 cm, and the quantitative counting zone is embedded with a pair of counter electrodes.

[0024] Furthermore, the electrode has a diameter of 2-2.5 mm, and the distances between the electrodes at both ends and the channel are equal. After being embedded, it is used to contact the working fluid in the flow channel and the droplets to be detected. The voltage between the opposing electrodes is 60-100 V, and the volume of the droplets to be detected is 6±1 μL.

[0025] The present invention also provides a method for detecting pathogenic microorganisms in a sample using the detection platform, comprising the following steps:

[0026] S1, such as Figure 2 The microbial detection platform is constructed as shown, and according to the functional requirements, the following are connected and constructed according to the positional relationship: a single-handed high-precision microfluidic pressure pump FLOW-EZ liquid pressure control unit (1), three Fluigent flow sensor monitoring units (2), three test tube liquid storage tanks (3), a waste liquid storage tank (4), a microfluidic chip body of the present invention (5), a chip fixture (6), a sample stock solution filtering valve (7), a dual-channel frequency phase-locked amplifier (8), a current amplifier (9), a wire (10), an inverted fluorescence microscope system (11), and a portable computer (12);

[0027] S2, chip channel flushing pretreatment, Figure 1 The organic buffer solution inlet of the chip body is injected with HFE 7500 buffer containing 5% wtsurfactant for 10-20 min. During this period, microscopic observation is used to ensure that impurities in the chip channel are washed away and the buffer solution flows evenly without bubbles.

[0028] S3, following step S2, debugging the frequency phase-locked amplifier, flow precision control unit, current amplifier, etc., and providing the buffer solution, immune nanomagnetic bead original body droplets, immune nanomagnetic bead droplets of pathogenic microorganisms to be detected, etc. by setting the optimal flow rate to generate standard reference signals with different impedance currents;

[0029] S4. Pretreatment of seawater samples. Since the in-situ extracted seawater sample solution contains many impurities, it needs to be filtered to remove large impurities that are easy to block the chip channel, and only retain 10-50μm particle samples, and control the sample concentration to 50-300 / μL;

[0030] S5. Adjust the parameter range of the detection platform. Set relevant instrument parameters according to the type of test sample and the test requirements, including the pressure control unit at the inlet of the buffer solution, sample stock solution, and immune nano-magnetic bead mixture, the flow monitoring unit, the amplification factor of the current amplifier, the frequency and amplitude of the lock-in amplifier excitation signal, the sampling frequency, the computer detection software mode, etc.;

[0031] S6. Use the pressure and flow precision control software AiO (full name Fluigent All-in-one) combined with the software of the phase-locked amplifier OE1022 to start the experiment. ① First pass the buffer solution - the flow monitoring is stable; ② Turn on OE1022, current amplifier, computer and other equipment to adjust the signal to be stable; ③ Pass the sample stock solution and immune nano-magnetic bead mixture to observe the droplet generation effect; ④ Start sampling the impedance corresponding current pulse signal;

[0032] S7. Analysis and processing of collected raw data: The collected raw impedance data - current pulse signal is filtered through the signal peak processing function in Labview to obtain the impedance peak characteristic signal of the sample to be tested.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The PDMS microchannel preparation method based on the ultra-slippery surface modification technology provided by the present invention can greatly improve the flux of liquid in the microchannel. Compared with the traditional method, it avoids the risk of secondary contamination or blockage caused by residual liquid samples in the channel. The operation steps are simple, and the functional surface has high stability and durability.

[0035] 2. The present invention improves the sorting efficiency by combining the prepared immune nanomagnetic beads with the magnetic control sorting function.

[0036] 3. The present invention adopts the Coulter counting technology of labeled fish pathogenic microorganisms, and uses the Coulter counting method of instantaneous fluctuation pulses of current generated by conductivity changes to achieve quantitative detection of fish pathogenic microorganisms.

[0037] 4. Compared with traditional laboratory incubation methods, microscope counting methods, etc., the present invention has the advantages of less sample consumption and shorter time consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of a magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with a super-lubricating surface according to an embodiment of the present invention.

[0039] Figure 2 It is a schematic diagram of the construction and use of the detection platform of the present invention.

[0040] Figure 3It is a schematic diagram of a PDMS (polydimethylsiloxane) microchannel based on the ultra-slippery surface modification technology of the present invention.

[0041] Figure 4 The PDMS@SiO 2 Contact angle diagram of a droplet on a super-slippery surface of self-assembled long chains on a substrate.

[0042] Figure 5 The PDMS@SiO 2 Rolling angle diagram of a droplet on a superslippery surface of substrate self-assembled long chains.

[0043] Figure 6 The present invention has PDMS@SiO 2 Diagram of droplet transport in a channel on a superslippery surface of self-assembled long chains on a substrate.

[0044] Figure 7 It is a schematic diagram of the process of preparing the immune nanomagnetic beads of the present invention and binding to target microorganisms.

[0045] Figure 8 It is a schematic diagram of the magnetic control separation function and actual effect diagram of the present invention.

[0046] Fig. 9 This is a graph of instantaneous fluctuation pulse signals of current generated by the immunonanomagnetic bead proto-droplets of the present invention.

[0047] Fig.10 It is a graph of instantaneous fluctuation pulse signals of current generated by the immune nanomagnetic bead droplets of the pathogenic microorganisms to be detected in the present invention. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0049] The experimental methods or test methods described in the following examples are conventional methods unless otherwise specified; the raw materials and auxiliary agents are obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0050] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0051] In some embodiments, a magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface comprises a PDMS microfluidic chip body;

[0052] Preferably, the PDMS microfluidic chip body is as follows Figure 1 The device shown includes: an organic buffer inlet (1), a sample solution inlet (2), a magnetic bead antibody mixture inlet (3), a mixed reaction area (4), a qualitative magnetic control sorting area (5), an enrichment area (6), a quantitative counting area (7) and a waste liquid outlet (8).

[0053] In some embodiments, the mixed reaction zone is PDMS@SiO 2 Substrate self-assembled long-chain superlubricating microchannels.

[0054] Preferably, the PDMS@SiO 2 The preparation method of the substrate self-assembled long-chain superlubricating microchannel comprises:

[0055] S1. Adding silica nanoparticles to toluene, stirring for 45-60 minutes under ultrasonic conditions to obtain a mixed solution in which the silica nanoparticles are fully dispersed in toluene;

[0056] S2, PDMS (polydimethylsiloxane, Sylgard 184) and the mixed liquid were stirred under ultrasonic conditions for 60-75 minutes, and then heated to completely volatilize the toluene, and then mixed with a curing agent (Dow Corning SYLGARD 184 SILICONE ELASTOMER KIT, USA) at a mass ratio of 7:1-9:1 to obtain a mixture, and then the mixture was degassed, and then the degassed mixture was poured into a mold and heated at 85-100°C for 1-1.5 hours to form PDMS@SiO 2 substrate;

[0057] S3, PDMS@SiO 2 The substrate was placed in an alkaline ethanol solution with a pH of ≥ 8.5 for 45-60 minutes to allow the SiO 2 The surface of the particles was fully hydroxylated, and then the PDMS@SiO 2 After the substrate material is completely air-dried in a fume hood, use plasma to modify the substrate material for 5-10 minutes (O 2 Concentration 65%) produces active hydroxyl functional groups on its surface;

[0058] S4, after treatment of PDMS@SiO 2 The substrate surface was sprayed with a mixture of toluene and DCDMS for hydrolysis reaction for 45-60 min, and then repeatedly rinsed with toluene solution for 10-15 min. After drying at room temperature (25±1°C, standard atmospheric pressure, ambient humidity 50%-65%), PDMS@SiO 2 Substrate self-assembled long-chain superlubricating microchannels.

[0059] More preferably, the SiO 2 The mass ratio of nanoparticles, PDMS and toluene is 5:8:87.2-8:10:87.2; more preferably, the SiO 2 The mass ratio of nanoparticles, PDMS and toluene is 7:10:87.2.

[0060] In some embodiments, the qualitative magnetic control sorting zone includes capturing pathogenic microorganisms using immunonanomagnetic beads.

[0061] In some embodiments, the method for preparing the immunonanomagnetic beads comprises the following steps:

[0062] S1. First, seal one end of the low protein adsorption centrifuge tube with 2%-3% BSA;

[0063] S2, washing the magnetic beads with PBS buffer and performing magnetic separation, removing the waste liquid, and then placing the magnetic beads in a low protein adsorption centrifuge tube, adding a certain amount of PBS buffer to re-dissolve, and then adding a certain amount of antibody to the low protein adsorption centrifuge tube and mixing with the magnetic beads, and then using PBS buffer to make the volume to 200 L, placing in a mixer, and reacting at room temperature at 35 rpm for 30-45 minutes;

[0064] S3, magnetically separating the reaction mixture until the magnetic beads are attached to the tube wall, and removing the remaining liquid;

[0065] S4, add PBS buffer to suspend the magnetic beads, then put them into the magnetic separator, let them stand until the antibodies connected to the magnetic beads are attached to the tube wall, and then suck away the remaining liquid again. Repeat this operation several times until the immune nanomagnetic beads are completely separated;

[0066] S5. Add PBS to suspend the immune nano-magnetic beads, and place in a refrigerator at a constant temperature of 4 degrees Celsius to obtain immune nano-magnetic beads.

[0067] In some embodiments, the quantitative counting area uses a Coulter counting method that uses conductivity changes to generate instantaneous fluctuation pulses of current to achieve quantitative analysis and detection of pathogenic microorganisms in the sample.

[0068] In some embodiments, the width of the channel from the organic buffer inlet to the mixing reaction zone is 50±5 μm, preferably 50 μm.

[0069] In some embodiments, the channel width of the mixed reaction zone is 50±5 μm, preferably 50 μm, and the length is 3-4 cm, preferably 4 cm, to ensure that the mixed reaction efficiency is not less than 90%.

[0070] In some embodiments, the channel width of the qualitative magnetic control sorting zone is 100±10 μm, preferably 100 μm, and the length of the strong magnetic zone is set to 1.5-2 cm, preferably 2 cm, to ensure that the magnetic field force can smoothly separate the immunomagnetic bead precursors, pathogenic microorganism immunomagnetic bead markers and non-magnetic particles in the "Y"-shaped sorting section.

[0071] In some embodiments, the quantitative counting area is embedded with a pair of counter electrodes, the electrode diameter is 2-2.5 mm, preferably 2 mm, the distance between the electrodes at both ends and the channel is equal, and after embedding, it is used to contact the working fluid in the flow channel and the droplets to be tested. The voltage between the counter electrodes is 60-100V, and the volume of the droplets to be tested is 6±1μL.

[0072] In some embodiments, a magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface includes a PDMS microfluidic chip body, a bonded packaging glass sheet, a liquid pressure control unit, a flow sensor monitoring unit, a test tube liquid storage tank, a waste liquid storage tank, a chip fixture, a sample liquid filter valve, a dual-channel frequency phase-locked amplifier, a current signal amplifier, a wire, an inverted fluorescence microscope system, a portable computer processor, a chip embedded electrode, a lead, and a display part.

[0073] According to the detection function requirements, such as Figure 2 The microbial detection platform is constructed as shown: a small air compressor is used to deliver air source to a single-hand-operated high-precision microfluidic pressure pump FLOW-EZ. The microfluidic pressure pump FLOW-EZ serves as a liquid pressure control unit (1) to push the buffer solution, sample solution and immune nanomagnetic bead mixture in three test tube storage tanks (3) into the microfluidic chip body (5) required by the present invention through the Fluigent flow sensor monitoring unit (2). In order to control the size of the detection particles, a sample solution filter valve (7) needs to be added between the sample solution and the flow sensor monitoring unit (2).

[0074] like Figure 2 As shown, the microfluidic chip body (5) and the chip fixture (6) of the present invention are both on the stage of the inverted fluorescence microscope system (11) to observe the sample generation during the overall detection process, which mainly includes chip channel flushing pretreatment, Figure 1 The organic buffer solution inlet of the chip body shown in the figure injects HFE 7500 buffer containing 5%wt surfactant for 10-20min. During this period, microscopic observation is used to ensure that the impurities in the chip channel are washed away and the buffer flows evenly without bubbles. The sample droplet generation is detected and the Figure 1 The sample stock solution and the immune nanomagnetic bead mixture are introduced into the main body of the chip, and the droplet generation effect is observed.

[0075] The phase-locked amplifier (8), the current amplifier (9) and the microfluidic chip body (5) on the microfluidic chip fixture (6) form an electrical circuit. The quantitative counting area is embedded in the counter electrode and the microfluidic channel area to form an impedance Coulter counting sensing area. The customized wire (10) connects the interdigitated counter electrode with the phase-locked amplifier (8) and the current amplifier (9) to form a detection circuit. The counter microelectrode is located on the upper part of the glass sheet bonded to the microfluidic chip by the sputtering method. After being embedded, it is used to contact the working fluid and the droplets to be detected in the flow channel. The electrode insertion direction is perpendicular to the direction of fluid flow and is used to conduct the response signal and detect the electrical impedance signal generated by the sample. By demodulating and analyzing the response current pulse signal, it is possible to realize the analysis of the composition, dielectric properties and number of the detected sample.

[0076] The detection platform also includes a waste liquid storage tank (4), a waste liquid storage tank (4) and Figure 1 The waste liquid outlet (8) in the microfluidic chip is connected to receive the waste liquid flowing out of the sample outlet channel in the microfluidic pipeline.

[0077] In some embodiments, the chip body manufacturing process is prepared using conventional techniques in the art such as soft lithography + overlay technology, and the chip channel is based on Figure 1 The width of the functional area is 50μm and 100μm respectively. SU8-2050 photoresist is used, and the average channel depth is controlled to be 60±10μm through the steps of coating, exposure, and development.

[0078] In some embodiments, the mixing reaction zone is a wavy microchannel. Preferably, the mixing reaction zone is a wavy 180° rotating microchannel to promote the sufficient mixing and reaction of the immune nanomagnetic bead mixture and the target pathogenic microorganism stock solution to achieve the function of specifically marking pathogenic microorganisms. Further preferably, the mixing reaction zone is a wavy 180° rotating microchannel prepared by a PDMS microchannel preparation method based on superlubricious surface modification technology.

[0079] The PDMS microchannel based on the superlubricious surface modification technology has a simple preparation process, high functional surface stability and strong durability. Figure 3 shown.

[0080] The base material of the PDMS microchannel is prepared by mixing PDMS (polydimethylsiloxane, Dow Corning 184, SYLGARD 184 SILICONE ELASTOMER, Sigma Aldrich supplier) doped with hydrophobic nano-silica particles (nano-silica particles with a particle size of 10-20 nm, purchased from Sigma Aldrich supplier) and a curing agent (type: Dow Corning SYLGARD 184 SILICONE ELASTOMER KIT, USA) in a mass ratio of 7:1-9:1, and after degassing, the mixture is evenly spread on a pre-prepared Figure 1 On the silicon wafer substrate with the chip microchannel pattern, the base material of the PDMS microchannel is formed after heating and curing at 85°C and demolding.

[0081] Then, a lubricating layer was self-assembled on the surface of the base material of the PDMS microchannel through modification technology to improve the liquid flux.

[0082] The self-assembled lubricating layer comprises two steps, namely, firstly forming a PDMS@SiO2 doped with hydrophobic nano-silica particles; 2 The substrate surface is then bonded to SiO 2 Long chain structures are grafted onto the particle surface.

[0083] The PDMS microchannel preparation method based on super lubricity surface modification technology comprises the following steps:

[0084] S1, PDMS@SiO 2 Preparation of substrate:

[0085] S1.1, adding silica nanoparticles into toluene solvent (purity 98%, Aladdin supplier), and magnetically stirring for 45 minutes under ultrasonic environment to make the silica nanoparticles evenly dispersed in the toluene solvent;

[0086] S1.2, then add PDMS, continue to magnetically stir for 60 minutes under ultrasonic environment to obtain a mixture, and magnetically stir and heat the mixture in a fume hood to remove all toluene;

[0087] S1.3, mix the mixed solution after removing all the toluene with the curing agent in a mass ratio of 7:1, stir thoroughly until the mixture is uniform, and then place it in a vacuum oven to remove bubbles;

[0088] S1.4, pour the mixture after removing bubbles into the mold and heat it at 100℃ for 1.5 hours to obtain PDMS@SiO 2 substrate;

[0089] S2, PDMS@SiO 2 Preparation of self-assembled lubricating layer on substrate surface:

[0090] S2.1, the PDMS@SiO 2 The substrate was placed in an alkaline ethanol solution for 45 min to allow the SiO 2 The surface of the particles is fully hydroxylized, and then the PDMS@SiO 2 The substrate was air-dried in a fume hood. The PDMS@SiO 2 The substrate was modified by plasma for 3 min (O 2 The modified substrate was sprayed with a diluted mixture of toluene and DCDMS (dichlorodimethylsilane, purchased from Shanghai MacLean Chemical Reagent Co., Ltd.) for a hydrolysis reaction for 45 min, so that the Si-Cl bonds and Si-(OH) bonds in the DCDMS reacted fully in the toluene environment, and finally formed a long-chain structure with Si-O-Si bonds as the main link. The substrate was then rinsed repeatedly with toluene solvent for 15 min to remove the reaction residues and finally retain the stable long-chain structure. After standing and drying at room temperature, a PDMS microchannel based on superlubricity surface modification technology with lubrication and effectiveness for up to 3 months was obtained.

[0091] In some embodiments, the pH of the alkaline ethanol solution is ≥ 8.5.

[0092] The PDMS@SiO 2 The effect of self-assembled long-chain ultra-slippery microchannels on the substrate was verified. Figure 4 , Figure 5 shown.

[0093] Depend on Figure 4 It can be seen that PDMS@SiO 2 The contact angles of the substrate self-assembled long-chain super-slippery material surface to water and oil droplets are both greater than 150°.

[0094] Depend on Figure 5 It can be seen that PDMS@SiO 2 The movement angle of droplets on the surface of substrate self-assembled long-chain superslippery materials is less than 10°.

[0095] The PDMS@SiO 2 Substrate self-assembled long chains on ultra-slippery microchannel surfaces Figure 1 The droplet transport effect in the mixing reaction zone is as follows Figure 6 shown.

[0096] Depend on Figure 6It can be seen that under the condition of oil phase buffer HFE 7500 as the continuous phase background, the droplets formed by the mixed solution of the sample stock solution and the immune nanomagnetic beads are smoothly transported and fully mixed in the "S"-shaped channel, and the ultra-slippery microchannel surface prevents the droplets from being blocked and retained at the 180° turn.

[0097] In order to achieve the qualitative separation of specific fish pathogenic microorganisms, such as Aeromonas punctata, Vibrio parahaemolyticus and other Gram-negative bacteria, the present invention prepares immune nanomagnetic beads and combines them with Figure 1 The qualitative magnetic separation area in the sample can achieve qualitative separation effect. Figure 7 As shown, taking Vibrio parahaemolyticus immune nanomagnetic beads as an example, nanomagnetic beads treated with an appropriate amount of activator (EDC) are coupled with biotin-modified Vibrio parahaemolyticus antibodies under appropriate conditions to form corresponding immune magnetic beads. The magnetic bead mixture is fully mixed with the sample stock solution to be tested. Under appropriate conditions, the membrane protein antibodies carried by the nanomagnetic beads will specifically bind to the Vibrio parahaemolyticus strain, achieving the purpose of non-invasive labeling, thereby facilitating the subsequent enrichment of the target strain.

[0098] The present invention prepares the immune nanomagnetic beads and Figure 1 The magnetic control separation zone in the above-mentioned PDMS@SiO2 is combined with the magnetic control separation zone to improve the separation efficiency. The specific pathogenic microorganisms with immune nanomagnetic beads are subjected to magnetic control qualitative separation in the magnetic control separation zone. The "Y" type separation section uses the above-mentioned PDMS@SiO2 2 The substrate self-assembled long-chain ultra-slippery microchannel technology is used to reduce the channel blockage caused by surface sticky residual samples during the sorting process. The magnetically controlled sorting area guides the labeled pathogenic microorganisms or immune nanomagnetic beads to the enrichment area to be tested.

[0099] In some embodiments, the immune nanomagnetic beads, also known as antibody-magnetic bead crosslinks, are surface-modified EDC-activated magnetic beads with biotinylated membrane protein antibodies and corresponding avidin (streptomycin to which Gram-negative bacteria are more sensitive), that is, the immune nanomagnetic beads are prepared by connecting avidin and biotin.

[0100] The materials required for preparing immunonanomagnetic beads include:

[0101] 1. 100 nm immunomagnetic beads (supplied by R&D System, USA, with Fe3O4 as the main component) with EDC-activated carboxyl groups linked to streptavidin were prepared at a concentration of 2 mg / ml;

[0102] 2. Biotin-labeled Gram-negative bacteria (Vibrio parahaemolyticus) (supplied by Biodesign International, Saco, ME, USA);

[0103] 3. Phosphate buffered saline (PBS, 0.01 M, pH 7.4) and bovine serum albumin (BSA) (supplied by Sigma-Aldrich, www.sigmaaldrich.com).

[0104] The preparation method of the immune nanomagnetic beads comprises:

[0105] S1. First, seal the low protein adsorption centrifuge tube with 3% BSA;

[0106] S2, wash the magnetic beads with 200 μL PBS, remove the waste liquid after magnetic separation on a magnetic separator, add a certain amount of PBS to re-dissolve, add a certain amount of antibody (0.4-0.6 mg / mL) to a centrifuge tube and mix with the magnetic beads, make up to 200 L with PBS buffer, put it in a mixer, and react at room temperature at 20 rpm for 30 min;

[0107] S3, placing the reaction mixture in a magnetic separator for magnetic separation until the magnetic beads are attached to the tube wall, and removing the remaining liquid with a pipette;

[0108] S4, add 200 μL of PBS buffer to suspend the magnetic beads, then put them into the magnetic separator, let them stand until the antibodies connected to the magnetic beads are attached to the tube wall, and then suck away the remaining liquid again. Repeat this operation several times until the immune nanomagnetic beads are completely separated;

[0109] S5. Add 30 μL of PBS to suspend the immune nano-magnetic beads, and place in a refrigerator at a constant temperature of 4 degrees Celsius to obtain the immune nano-magnetic beads.

[0110] Magnetic separation function diagram and actual effect Figure 8 shown.

[0111] Depend on Figure 8 It can be seen that after the droplets formed by the sample stock solution and the mixed solution of the immune nanomagnetic beads are fully mixed and reacted, the membrane protein antibodies carried by the immune nanomagnetic beads will specifically bind to the Vibrio parahaemolyticus strain. In the "Y"-shaped channel, due to the action of the magnetic field force below, (a) droplets containing immune nanomagnetic beads and (b) droplets of immune nanomagnetic beads combined with the pathogenic microorganisms to be tested are pulled into the channel below.

[0112] In order to achieve quantitative detection of pathogenic microorganisms, the present invention applies the Coulter technique principle in the quantitative counting area, uses embedded electrodes in the flow channel, and combines Figure 1 When the droplet to be tested passes through the quantitative counting area from the enrichment area to the end of the chip, the object to be tested or the immune nanomagnetic bead body is marked, and the conductivity changes through the electrode end, resulting in different instantaneous fluctuation pulse signals of the current, thereby realizing the quantitative analysis function.

[0113] Fig. 9 What is shown is the instantaneous fluctuation pulse signal of the current in the droplet containing a large number of free immune nano-magnetic beads without binding the pathogenic microorganisms to be detected.

[0114] Fig.10 Shown is the instantaneous fluctuation pulse signal of the current in the droplet containing the immune nanomagnetic beads combined with the pathogenic microorganisms to be detected.

[0115] The present invention screens the sampled data through the signal peak processing function in Labview, obtains the impedance peak characteristic signal of the sample to be tested, and samples and analyzes the instantaneous fluctuation pulse signal of the current generated by the conductivity (impedance) change, so that the counting effect is more accurate.

[0116] Application Examples

[0117] The method for detecting the pathogenic microorganism Gram-negative bacteria-Vibrio parahaemolyticus in a sample using the detection platform has the following specific steps:

[0118] S1. Pre-processing of the microfluidic chip Figure 1 The organic buffer solution inlet of the chip body is injected with HFE 7500 buffer containing 5%wt surfactant for 10-20min, during which microscopic observation is used to ensure that impurities in the chip channel are flushed clean and the buffer flows evenly without bubbles.

[0119] S2. Optimize the parameter acquisition method, perform spectrum scanning on the target detection object between the Coulter counting detection electrodes, set the single buffer OT1, immune nanomagnetic bead original body droplet OT2, and immune nanomagnetic bead droplet OT3 of the pathogenic microorganism to be detected as reference extraction signals in the parameter setting of the phase-locked amplifier, select the frequency scanning range of 0.8kHz-5MHz, and finally determine that the frequency of the excitation pulse electrical signal of the immune nanomagnetic bead droplet of different sizes of Vibrio parahaemolyticus to be detected is composed of the superposition of several frequencies such as 1000kHz and 3.5MHz, 500kHz and 5MHz;

[0120] S3. Preparation of Vibrio parahaemolyticus immune nanomagnetic beads. The cells used in the experiment were Gram-negative bacteria - Vibrio parahaemolyticus (supplied by Biodesign International, Saco, ME, USA). The parameters of the biological constant temperature incubator used were set as follows, and the incubator temperature was kept constant at 37°C. BSA culture was used, and the concentration of the suspension prepared by Vibrio parahaemolyticus immune nanomagnetic beads was controlled at 250-300 / μL in the experiment;

[0121] S4. Other relevant parameters of the detection platform are set. The injection pressure of the cell sample is 200-500 mbar, the injection flow rate is controlled at 3-10 μL / min, the amplification factor of the transimpedance current amplifier is set to 1000 times, the sampling frequency is set to 224.9 Sa / s, and the impedance detection mode setting of the lock-in amplifier is preferably set;

[0122] S5. Test results Fig. 9 , Fig.10 As shown in the figure, the impedance pulse signal differences of different types of droplets to be tested are shown. Since the impedance pulse signal characteristics and distributions generated by different types of droplets to be tested are obviously different, it is confirmed that the constructed electrical impedance Coulter counting detection platform can be used to obtain non-invasive and label-free parameters for distinguishing fish pathogenic microorganisms.

[0123] Aiming at the needs of qualitative detection and quantitative counting of microbial pathogens in complex seawater samples, the present invention proposes to develop PDMS@SiO 2 The present invention provides technical support for the rapid detection of microbial pathogens in marine ranch ecosystems, including the ultra-slippery surface of self-assembled long chains of substrates, the magnetic control sorting function of immune nanomagnetic beads, and the Coulter counting method of instantaneous fluctuation pulses of current generated by changes in conductivity of different liquid phases.

[0124] The present invention first utilizes PDMS@SiO 2 The substrate self-assembles into a long-chain ultra-slippery surface to reduce biological adhesion and sample loss, thereby improving the detection accuracy of the system. The magnetically controlled separation and sorting function of the immune nanomagnetic beads is then used to capture and enrich pathogenic microorganisms in complex samples. The Coulter counting method, which uses instantaneous fluctuation pulses of current generated by changes in conductivity, is then used to quantitatively count the captured samples, ultimately achieving the detection of pathogenic microorganisms in seawater samples on the chip.

[0125] The research results of this invention will promote the development and transformation of microfluidic technology in the field of marine aquaculture microbial pathogen detection, and provide a theoretical basis and technical support for improving the accuracy, efficiency and automation of LOC technology in rapid pathogen detection.

[0126] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface, characterized in that: The detection platform includes a PDMS microfluidic chip body; The PDMS microfluidic chip body comprises: an organic buffer inlet (1), a sample stock solution inlet (2), a magnetic bead antibody mixed solution inlet (3), a mixed reaction area (4), a qualitative magnetic control sorting area (5), an enrichment area (6), a quantitative counting area (7) and a waste liquid outlet (8); The mixing reaction zone (4) is a PDMS@SiO2 substrate self-assembled long-chain super-lubricating microchannel; The preparation method of the PDMS@SiO2 substrate self-assembled long-chain superlubricating microchannel comprises: S1, adding SiO2 nanoparticles into toluene and stirring evenly to obtain a mixed solution in which SiO2 nanoparticles are fully dispersed in toluene; S2, after the PDMS and the mixed solution are stirred evenly, toluene is removed, and then the mixture is mixed with the curing agent in a mass ratio of 7:1-9:1 to obtain a mixture, and then the mixture is defoamed, and then the defoamed mixture is poured into a mold to form a PDMS@SiO2 substrate; S3, immersing the PDMS@SiO2 substrate in an alkaline ethanol solution with a pH value of ≥ 8.5, then taking out the PDMS@SiO2 substrate dipped in the ethanol residue and air-drying it, and then using plasma to modify the air-dried substrate; S4. Spray a mixture of toluene and DCDMS on the surface of the modified PDMS@SiO2 substrate to perform a hydrolysis reaction, then rinse repeatedly with toluene solution, and after drying at room temperature, obtain a self-assembled long-chain superlubricating microchannel on the PDMS@SiO2 substrate.

2. The magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface according to claim 1, characterized in that: The mass ratio of the SiO2 nanoparticles, PDMS and toluene is 5:8:87.2-8:10:87.

2.

3. The magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface according to claim 1, characterized in that: In step S2, the temperature of the mold is 85-100° C. and the mold is heated for 1-1.5 hours.

4. The magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface according to claim 1, characterized in that: The immersion time in step S3 is 45-60 minutes, the modification time in step S3 is 5-10 minutes, and the O2 concentration during the modification process is 65%.

5. The magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface according to claim 1, characterized in that: The hydrolysis reaction time in step S4 is 45-60 min.

6. The magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface according to claim 1, characterized in that: The qualitative magnetic control sorting area includes capturing pathogenic microorganisms using immune nanomagnetic beads.

7. The magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface according to claim 1, characterized in that: The quantitative counting area uses a Coulter counting method that uses conductivity changes to generate instantaneous fluctuation pulses of current to achieve a quantitative analysis and detection function for pathogenic microorganisms in a sample.

8. The magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface according to claim 1, characterized in that: The width of the channel from the organic buffer inlet to the mixed reaction zone is 50±5 μm, the width of the channel in the qualitative magnetic control sorting zone is 100±10 μm, and the length is 1.5-2 cm. The quantitative counting zone is embedded with a pair of counter electrodes.

9. The magnetically controlled fish pathogenic microorganism microfluidic chip detection platform with an ultra-slippery surface according to claim 8, characterized in that: The electrode diameter is 2-2.5 mm, the distance between the electrodes at both ends and the channel is equal, and after being embedded, it is used to contact the working fluid in the flow channel and the droplet to be detected. The voltage between the opposing electrodes is 60-100 V, and the volume of the droplet to be detected is 6±1 μL.

Images