A charge-mediated microchannel current biosensing method based on directional coupling technology and its application

Abstract

The present invention relates to a charge-mediated microchannel current biosensing method based on directional coupling technology and its application. By pre-modifying the inner surface of a microchannel with a click reagent 1 efficiently, adding a biorecognition molecule coupled with a click reagent 2 when to be detected, inducing a click reaction so that the biorecognition molecule is directional coupled and fixed on the inner wall of the microchannel. The target to be detected and the self-assembled protein spheres containing detection antibodies and bovine serum albumin are added in sequence to generate an immune capture reaction. The protein spheres fixed on the inner surface of the microchannel reaction area will cause the charge density of the inner surface of the microchannel to change, and the content of the target to be detected can be indirectly obtained by detecting the change in the current or voltage of the closed-loop circuit. The present invention utilizes directional coupling technology and combines the assembly scheme of click chemistry reaction, which can effectively solve the problem of biochemical activity decay of biorecognition molecules during long-term storage, while avoiding complex enzymatic reactions, and further improving the stability of the method.

Description

Technical Field

[0001] The invention belongs to the field of detection, and specifically relates to a microchannel biosensing method based on directional coupling technology and charge density mediation and an application thereof. Background Art

[0002] In response to the needs of fast, accurate and high sensitivity detection in the fields of food safety and disease diagnosis, biosensors with different mechanisms have emerged and developed rapidly. Among them, improving the signal conversion mechanism and monitoring mode has become an important means to improve the sensitivity and stability of detection. In the early stage, the inventor's research group used polymer microspheres as solid phase matrix to construct a biosensor detection method based on the change of microchannel current caused by the concentration / state change of insulating microspheres and a matching biosensor detection device (CN 112415058 A, CN 112595759 A). By modifying the biorecognition molecules on the surface of solid phase microspheres (magnetic beads or insulating microspheres), adding the target to be tested for biological reaction, the solid phase microsphere complex is subjected to magnetic separation or directly used for electrical signal detection of the biosensor detection device, which has good analytical performance, low equipment cost, simple and efficient, and has good application prospects. However, labeling biorecognition molecules with polymer microspheres as solid phase matrix not only increases the complexity of operation and the analysis cost, but also the self-aggregation between polymer microspheres will make it difficult to ensure the accuracy of the detection signal, which is not conducive to the further application of this method. Based on this, in order to achieve better universality and wider application of the constructed biosensing method, further research is needed to reduce economic and time costs and improve accuracy and stability. Summary of the invention

[0003] The purpose of the present invention is to solve the problems of complex operation, high cost, poor accuracy, complex detection mechanism, and easy occurrence of false results in existing electrical signal biosensor methods and detection devices, and to provide a microchannel biosensor method based on directional coupling technology and charge density mediation and its application. This method has outstanding advantages such as fast response speed, low economic cost, and good stability, and has the potential to be widely used in the rapid detection of various hazardous factors in food, clinical and environmental conditions.

[0004] The above objectives are mainly achieved through the following technical solutions:

[0005] A charge-mediated microchannel current biosensing method based on directional coupling technology, the method comprising the following steps:

[0006] S1 pre-modifies the click reagent 1 on the inner surface of the microchannel, and when testing, adds the biorecognition molecule coupled with the click reagent 2, induces the click reaction, and makes the biorecognition molecule fixed on the inner wall of the microchannel by directional coupling, and sequentially adds the target to be tested and the self-assembled protein ball containing the detection antibody and bovine serum albumin for immune capture reaction. The protein ball can efficiently amplify the signal of each antigen-antibody specific binding reaction. The charge state of the inner surface of the microchannel changes, and the change in charge density is related to the concentration of the target to be tested;

[0007] The S2-modified microchannel is fixed in the middle of the two conductive liquid pools, becoming the only channel connecting the two conductive liquid pools. The positive and negative electrodes are respectively inserted into the conductive cells at both ends of the microchannel and a constant voltage or current is applied, forming a closed-loop circuit between the electrode-electrolyte-microchannel.

[0008] The S3 sample solution passes through the microchannel, undergoing a specific biorecognition reaction, and then self-assembled protein spheres containing detection antibodies and bovine serum albumin are added. Since the protein surface carries a negative charge, the aggregation of proteins into protein spheres can cause a greater change in the charge density in the microchannel, which in turn causes a change in the current of the closed-loop circuit. The degree of change is related to the charge density in the microchannel, so that the content of the target substance to be measured can be obtained.

[0009] Preferably, in step S1, click reagent 1 and click reagent 2 include tetrazine group (TZ)-trans-cyclooctene (TCO), azide-alkyne, dibenzocyclooctyne (DBCO)-azide (Azide), cysteine ​​(Cys)-cyanobenzothiazole (CBT) or others.

[0010] Preferably, the biorecognition molecule is an antibody or antigen that can undergo an immune reaction with the target to be detected, or the biorecognition molecule is a bacteriophage that can specifically capture microorganisms, or the biorecognition molecule is a nucleic acid aptamer of a capture probe that can undergo a DNA molecule hybridization reaction with the target to be detected, or the biorecognition molecule is a nanobody that specifically captures the target to be detected.

[0011] Preferably, the inner diameter of the microchannel is 25-75 μm, and the length is 0.5-5 mm. The inner diameter of the microchannel is 50 μm, and the length is 3 mm.

[0012] Preferably, the concentration of the biorecognition molecules modifying the inner wall of the microchannel is 1 μg / mL~30 μg / mL, and the optimal concentration is 20 μg / mL; when a constant voltage is applied to the positive electrode and the negative electrode, the voltage is 50-300 V, and the optimal concentration is 100 V; the electrolyte is 0.1 mol / L KCl salt solution.

[0013] Preferably, in step S1, the microchannel is a capillary, and the biorecognition molecules are modified on the inner surface of the microchannel by directional coupling technology, and the specific operation includes the following steps:

[0014] S1 A.1 Rinse the capillary with hydrochloric acid to remove contaminants adhering to the inner wall;

[0015] S1 A.2 Continue to rinse the capillary with sodium hydroxide to activate the silanol groups on the inner wall of the capillary;

[0016] S1A.3 Take the surface-immobilized atom transfer radical polymerization initiator [11-(2-bromo-2-methyl)propionyloxy] undecyltrichlorosilane (5 wt%, dissolved in toluene) and inject it into the capillary microchannel for reaction for 2 h. After the reaction, the microchannel is cleaned with toluene and methanol in turn;

[0017] S1A.4 Silicon-based block graft copolymerization: 2-methacryloyloxyethyl phosphorylcholine, copper bromide and 2,2'-bipyridine were added to degassed methanol, and the mixed solution was injected into the capillary microchannel for polymerization for 2 h. After the polymerization reaction was completed, CuBr2 solution was injected into the microchannel to make the inner surface of the microchannel a bromide ion end group, thus completing the block copolymerization.

[0018] S1A.5 Add N-(2-aminoethyl)methacrylamide hydrochloride, copper bromide and 2,2'-bipyridine to a mixed solution of isopropanol and water (isopropanol / water = 8:2 v / v), and stir the mixed solution with [11-(2-bromo-2-methyl)propionyloxy]undecyltrichlorosilane;

[0019] S1A.6 After incubating the tyrosinase solution and the Staphylococcus aureus protein A solution at room temperature, inject them into the capillary microchannel and react at room temperature;

[0020] S1A.7 Dissolve TCO in DMF, inject into the capillary microchannel and incubate at room temperature for 1 h. After the reaction is complete, wash with PBST, add 5% BSA and block at 37℃ for 30 min. After washing with PBST, store at 4℃ for later use.

[0021] S1 A.8 Dissolve TZ in DMF, mix TZ and biorecognition molecules at room temperature and stir for 1 h. After the reaction is completed, add Tris-HCl to the mixture to terminate the reaction for 10 min. Add the mixture to a dialysis bag and dialyze at 4 °C for 12 h.

[0022] Finally, the prepared TZ-biorecognition molecule conjugate was stored at -20°C for later use; when it was to be detected, it was injected into the capillary microchannel, and the modification process was completed at room temperature for 15 minutes.

[0023] Preferably, the self-assembled protein spheres in step S1 include detection antibodies and bovine serum albumin, horseradish peroxidase, alkaline phosphatase or others; the protein sphere self-assembly process includes the following steps:

[0024] S1B.1 Mix 450 μL of bovine serum albumin solution (20 mg mL −1 )、50uL detection antibody (20 mg mL −1 ) and 500 µL of the surfactant CTAB (20 mg mL −1 ), the three synthesized protein nanoconjugates through electrostatic reaction;

[0025] S1B.2 Add 60 µL of isooctyl alcohol to the aqueous solution of the nanoconjugate to form a protein ball dispersion. Then add 10 µL of glutaraldehyde (20%) to react the exposed amines with glutaraldehyde to crosslink the protein molecules in the surface membrane of the protein ball, and react at the water / oil interface for 4 h;

[0026] S1B.3 The protein spheres are dialyzed against a water / ethanol mixture to remove isooctyl alcohol and CTAB to ensure that the protein spheres are transferred to the aqueous phase without membrane collapse.

[0027] The proteins (bovine serum albumin and detection antibody) prepared by the above method have dual functions: specific capture ability of target antigen / antibody and electrical signal amplification function due to higher protein loading. Taking bovine serum albumin sphere as an example, bovine serum albumin isoelectric point is 4.7, and the environmental pH (7.4) is higher than its isoelectric point pI and is negatively charged. Aggregating multiple bovine serum albumins into spheres can cause greater current changes in the system.

[0028] This method constructs a charge quenching theory based on immune recognition. When the antibody binds to the antigen, its positive charge is quenched, resulting in a decrease in the current. In order to improve the reaction sensitivity, a protein ball signal amplification system containing detection antibodies is introduced. The number of targets causes different numbers of detection antibodies specifically recognized on the inner surface of the microchannel, that is, different numbers of protein balls on the inner surface, resulting in different changes in the current.

[0029] A microchannel current biosensor monitoring device based on the change of the charge on the inner surface of a microchannel, the device includes an LED electronic touch screen, a fully automatic three-axis motion control component, a vacuum pressure device for sucking and injecting liquids, a reagent loading platform, two platinum wire electrodes with an outer diameter of 1.5 mm, and a fused quartz capillary microchannel; the bottom of the reagent storage bottle is hollow and connected to the microchannel limit, and the type and volume of the reagent injected into the microchannel are adjusted by the vacuum pressure device. A reagent tray and a rubber plug for preventing back suction and fixing are provided above the reagent storage bottle.

[0030] Preferably, the fully automatic three-axis motion control component controls the position of biochemical reagents, adjusts the volume of the solution in the hollow reagent storage bottle, and controls the switch of the test system power supply through the touch screen; the vacuum pressure system maintains a sealed state through a rubber stopper and the hollow reagent storage bottle fixed to the reagent loading platform to apply pressure to control the injection and removal of the solution; the reagent loading platform is an element for placing biochemical reagents used for chemical modification, biorecognition molecule labeling and sample detection; the two platinum wire electrodes are respectively immersed in the reagent disk and the reagent storage bottle, forming a closed loop with the microchannel fixed by the gasket; the microchannel is a solid phase carrier of the biorecognition molecule labeling; the power module provides a power source for the sensing detection device, the fully automatic three-axis motion control component and the pressure system; the electrical signal monitor transmits the detected electrical signal to the touch screen through the control and data conversion module to realize the collection and processing of the electrical signal.

[0031] A multi-channel microfluidic chip based on the change of surface charge density in a microchannel leading to the change of microchannel current, the chip is provided with a sample area, two conductive liquid pools and a microchannel; the sample area is located upstream of the microchannel conductive liquid pool to store samples to be tested, the two conductive liquid pools are connected through the microchannel and are respectively provided with a positive electrode and a negative electrode inside to form a closed-loop circuit; the closed-loop circuit is provided with a digitally controlled current monitor which can continuously monitor the current change.

[0032] Preferably, the inner diameter of the microchannel is 50-100 μm, the length is 0.5-5 mm, and the material is polystyrene (PS), polymethyl methacrylate (PMMA) or polydimethylsiloxane (PDMS).

[0033] Preferably, the charge-mediated microchannel current biosensing method based on directional coupling technology or the device is used to detect disease markers, antibiotics, pathogenic microorganisms, pesticides and veterinary drugs or others.

[0034] Preferably, different detection methods are used for different targets:

[0035] like Figure 2As shown in the figure, when the analyte is a macromolecule, such as procalcitonin, the click reagent TCO is injected into the microchannel in advance and allowed to stand at room temperature for 1 h to modify the click reagent on the inner surface. When the test is to be detected, the capture antibody coupled with the click reagent TZ is added to induce a click reaction so that the capture antibody is directionally coupled to the inner wall of the microchannel, and the target to be detected and the protein ball containing the detection antibody are added in sequence for immune capture reaction to form a sandwich complex, so that the charge state of the inner surface of the microchannel changes, and the change in charge density is positively correlated with the concentration of the target to be detected; when the analyte is a small molecule, such as chloramphenicol, the click reagent TCO is injected into the microchannel in advance and allowed to stand at room temperature for 1 h to modify the click reagent on the inner surface. When the test is to be detected, the complete antigen coupled with the click reagent TZ is added to induce a click reaction so that the complete antigen is directionally coupled to the inner wall of the microchannel, and the target to be detected and the capture antibody are added for competitive immune reaction, and then combined with the protein ball containing the detection antibody, so that the charge state of the inner surface of the microchannel changes, and the change in charge density is negatively correlated with the concentration of the target to be detected.

[0036] In this method, click reagent 1 is injected into the microchannel in advance and allowed to stand at room temperature for 1 hour to modify the inner surface. When the detection is to be performed, biorecognition molecules (antibodies, antigens, bacteriophages, nucleic acid aptamers, and nanoantibodies) coupled with click reagent 2 are added to induce a click reaction so that the biorecognition molecules are directional coupled and fixed on the inner wall of the microchannel, achieving the purpose of efficient modification and specific capture of the target. Since the introduction of the target and the protein ball containing the detection antibody will cause the charge density on the inner surface of the microchannel to change, different concentrations of the captured target will produce different current signal responses under constant voltage, and the current is quantitatively analyzed as a readout signal by real-time monitoring of the current monitoring device.

[0037] The present invention utilizes directional coupling technology and combines the assembly scheme of click chemistry reaction, which can effectively solve the problem of biochemical activity decay of biological recognition molecules during long-term storage. At the same time, based on the charge quenching theory of immune recognition and the electrical signal amplification effect of protein balls, it greatly improves the sensitivity of the method while improving the coupling efficiency, reduces the detection cost and experimental analysis time, has the advantages of accuracy, speed, stability, etc., and has good application prospects in fields that require on-site rapid detection, such as food safety, in vitro diagnosis, and environmental monitoring.

[0038] The beneficial effects of the present invention are:

[0039] 1) High analysis efficiency and fast analysis speed: The present invention adopts directional coupling technology and combines the assembly scheme of click chemistry reaction to fix the biorecognition molecules on the inner surface of the microchannel, which has high labeling and capture efficiency; and the entire analysis process of the target object can achieve high-throughput detection, which has the advantages of high analysis efficiency and fast analysis speed.

[0040] 2) Good stability and low detection cost: The readout signal of the present invention is a current signal, the readout device is simple and not easily affected by environmental factors, and the reaction area of ​​the present invention is the inner surface of a micron-scale microchannel, the reagent consumption is very small, the pre-coupled click reagent has good stability, the reagent utilization rate is high, and no complex instruments are required. Only a real-time monitoring current monitoring device is required, and the cost of the entire device is less than 300 yuan.

[0041] 3) Wide range of applications: By directionally coupling different biorecognition molecules to the inner surface of the microchannel, quantitative analysis of a variety of small molecules, nucleic acids, and pathogenic bacteria can be performed, which has potential application value in food safety, in vitro diagnosis, environmental monitoring and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Fabrication process diagram of functionalized capillary microchannels.

[0043] Figure 2 : Schematic diagram of microchannel current biosensor device for different targets.

[0044] Figure 3 : Schematic diagram of the structure of microfluidic chip sensor.

[0045] Figure 4 : Schematic diagram of the structure of the integrated microchannel current biosensor device.

[0046] Figure 5 : Correlation between surface charge density and current.

[0047] Figure 6 The inner diameters of the microchannels were 25 μm, 50 μm, 75 μm, and 100 μm, respectively, and the length was 3 mm. The TCO coating concentration was 15 μg / mL, the TZ-procalcitonin capture antibody concentration was 20 μg / mL, the protein ball concentration containing the procalcitonin detection antibody was 20 μg / mL, and the procalcitonin concentrations were 0, 1, 10, and 10 μg / mL. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 pg / mL, applied voltage was 100 V, and the correlation between the measured current change value (ΔI) and the logarithm of procalcitonin concentration.

[0048] Figure 7The inner diameter of the microchannel was 50 μm, and the lengths were 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, and 5 mm, respectively. The TCO coating concentration was 15 μg / mL, the TZ-procalcitonin capture antibody concentration was 20 μg / mL, the protein ball concentration containing the procalcitonin detection antibody was 20 μg / mL, and the procalcitonin concentration was 0, 1, 10, and 10 μg / mL. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 pg / mL, applied voltage was 100 V, and the correlation between the measured current change value (ΔI) and the logarithm of procalcitonin concentration.

[0049] Figure 8 : The inner diameter of the microchannel is 50 μm, the length is 3 mm, the TCO coating concentrations are 5 μg / mL, 10 μg / mL, 15μg / mL, and 20 μg / mL, respectively, the TZ-procalcitonin capture antibody concentration is 20 μg / mL, the protein ball concentration containing procalcitonin detection antibody is 20 μg / mL, the applied voltage is 100 V, and the correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration.

[0050] Fig. 9 : The inner diameter of the microchannel is 50 μm, the length is 3 mm, the TCO coating concentration is 15 μg / mL, the TZ-procalcitonin capture antibody concentrations are 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL, respectively, the concentration of the protein ball containing the procalcitonin detection antibody is 20 μg / mL, the applied voltage is 100 V, and the correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration.

[0051] Fig.10 : The inner diameter of the microchannel is 50 μm, the length is 3 mm, the TCO coating concentration is 15 μg / mL, the TZ-procalcitonin capture antibody concentration is 20 μg / mL, the concentrations of the protein balls containing the procalcitonin detection antibody are 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL, respectively, and the applied voltage is 100 V. The correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration.

[0052] Fig.11: The inner diameter of the microchannel is 50 μm, the length is 3 mm, the TCO coating concentration is 15 μg / mL, the TZ-procalcitonin capture antibody concentration is 20 μg / mL, the protein ball concentration containing the procalcitonin detection antibody is 20 μg / mL, the applied voltage is 50 v, 100 v, 200 v, 300 v, and the correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration.

[0053] Fig.12 : The correlation between the current change value (ΔI) caused by the bioactivity decay of the biorecognition molecules directly coupled to the inner wall of the microchannel and the pre-coupled click reagents and the procalcitonin concentration.

[0054] Fig.13 : Correlation between the current change value (ΔI) measured by different protein ball-TZ conjugate and detection antibody-TCO conjugate concentration ratios and procalcitonin concentration when detecting procalcitonin.

[0055] Fig.14 : The signal amplification effect of adding protein beads containing detection antibodies and the correlation between the current change value (ΔI) caused by the one-step immune reaction and the procalcitonin concentration.

[0056] Fig.15 : Correlation between the current change value (ΔI) and procalcitonin concentration measured in different buffer solutions when detecting procalcitonin.

[0057] Fig.16 : Correlation between the current change value (ΔI) measured at different competition reaction times and the procalcitonin concentration when detecting procalcitonin.

[0058] Fig.17 : When detecting procalcitonin, the correlation between the current change value (ΔI) measured by the reaction time between protein balls containing different detection antibodies and procalcitonin and the procalcitonin concentration.

[0059] Fig.18 : Working curve for detecting procalcitonin.

[0060] Fig.19 : Correlation between the current change value (ΔI) measured by different concentrations of chloramphenicol antibodies and the chloramphenicol concentration when detecting chloramphenicol.

[0061] Fig. 20 : Correlation between the current change value (ΔI) measured by different concentrations of chloramphenicol capture antibodies and the chloramphenicol concentration when detecting chloramphenicol.

[0062] Fig.21 : Correlation between the current change value (ΔI) measured at different immune response times and the chloramphenicol concentration when detecting chloramphenicol.

[0063] Fig. 22 : Working curve for detecting chloramphenicol.

[0064] Fig.23 : Correlation between the current change value (ΔI) measured by different concentrations of Salmonella-coated antibodies and the Salmonella concentration when detecting Salmonella.

[0065] Fig.24 : Correlation between the current change value (ΔI) measured at different immune response times and the Salmonella concentration when detecting Salmonella.

[0066] Fig.25 : Working curve for detecting Salmonella. DETAILED DESCRIPTION

[0067] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0068] Test materials and related terms

[0069] Sodium hydroxide, toluene, cetyltrimethylammonium bromide (CTAB), isooctyl alcohol, glutaraldehyde, and ethanol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0070] [11-(2-Bromo-2-methyl)propionyloxy]undecyltrichlorosilane: purchased from Shanghai Merrill Chemical Technology Co., Ltd.

[0071] 2-Aminoethyl methacrylate: purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd.

[0072] 2-Methacryloyloxyethyl phosphorylcholine: purchased from Suzhou Yipeter Biotechnology Co., Ltd.

[0073] 2,2'-Bipyridine: purchased from Jinan World Chemical Co., Ltd.

[0074] Trans-cycloolefin (TCO-PEG4-NHSEster, TCO) and tetrazine group (Tetrazine-PEG4-NHSEster, TZ): purchased from Click Chemistry Tools.

[0075] Chloramphenicol antibody (1 mg / mL), chloramphenicol-BSA conjugate (1 mg / mL), and chloramphenicol standard were purchased from Henan Lufeifan Biotechnology Co., Ltd.

[0076] Procalcitonin (1.4 mg / mL), procalcitonin capture antibody (8.5 mg / mL), and procalcitonin detection antibody (8.35 mg / mL) were purchased from Beijing Anbaisheng Biotechnology Co., Ltd.

[0077] Salmonella: from bacteria preserved in the laboratory.

[0078] Quartz capillary: purchased from Handan Yongnian Ruifeng Chromatography Devices Co., Ltd.

[0079] A microchannel current biosensor monitoring device based on the change of surface charge in a microchannel, the device comprises a touch screen 1, a fully automatic three-axis moving control part 2, a pressure system 3, a reagent disk 4, two platinum wire electrodes 5, a microchannel 6, a power module, an electrical signal monitor, and a control and data conversion module. The bottom of a reagent bottle 9 is hollowed out and connected to the microchannel 6. The type of reagent injected into the microchannel is adjusted by the pressure control system 3. A reagent disk 7 and a rubber plug 10 are arranged above the reagent bottle 9. The purpose of the rubber plug is to ensure that the pressure control system is in a vacuum state so as to achieve liquid injection through negative pressure. The fully automatic three-axis moving control part 2 controls the pressure of the microchannel 6 through the touch screen 1. The position of biochemical reagents is regulated, the volume of the solution in the hollow reagent bottle 9 is adjusted, and the switch of the power supply of the test system is controlled, so as to integrate the preparation of functionalized microchannels and the detection of sample molecules and other automated functional operations; the pressure system 3 is kept sealed by the hollow reagent bottle 9 fixed by the rubber stopper 10 and the reagent disk 4, so as to apply a suitable pressure to control the injection and removal of the solution; the reagent disk 4 is a component for placing biochemical reagents used for chemical modification, biorecognition molecule labeling and sample detection; the two platinum wire electrodes 5 are immersed in the conductive liquid pool 7 and the reagent bottle 9 respectively, and form a closed loop with the microchannel 6 fixed by the gasket 8; the microchannel 6 is a solid phase carrier of the biorecognition molecule labeling; the power module provides a power source for the sensing detection device, the fully automatic three-axis motion control component and the pressure system; the electrical signal monitor transmits the detected electrical signal to the touch screen through the control and data conversion module to realize the collection and processing of the electrical signal.

[0080] Example 1 Preparation of functionalized capillary microchannels based on directional coupling technology

[0081] Directed coupling technology is combined with click reaction. By pre-modifying the click reagent 1 efficiently on the inner surface of the microchannel, when testing, only the biorecognition molecule coupled with the click reagent 2 needs to be added to induce the click reaction so that the biorecognition molecule is quickly and directionally coupled and fixed on the inner wall of the microchannel, and the coating process can be completed, which greatly improves the reaction efficiency. The present invention uses directed coupling technology to modify the biorecognition molecule on the inner wall of the quartz capillary to specifically capture the target. The modification reagent and the biorecognition molecule are injected into the capillary in turn under negative pressure through a fully automatic three-axis motion control system and a pressure system. The specific modification process is as follows:

[0082] (1) Rinse the capillary with 0.1 mol / L hydrochloric acid to remove any contaminants attached to the inner wall;

[0083] (2) Rinse with 0.1 mol / L sodium hydroxide at room temperature for 30 min to activate the silanol groups on the inner wall of the capillary;

[0084] (3) Take the surface-immobilized atom transfer radical polymerization initiator [11-(2-bromo-2-methyl)propionyloxy] undecyltrichlorosilane (5 wt%, dissolved in toluene) and inject it into the capillary microchannel for reaction for 2 h. After the reaction, the microchannel is cleaned with toluene and methanol in turn;

[0085] (4) Silicon-based block graft copolymerization. 7.00 mmol 2-methacryloyloxyethyl phosphorylcholine, 0.14 mmol copper bromide and 0.28 mmol 2,2'-bipyridine were added to 10 mL of degassed methanol, and the mixed solution was injected into the capillary microchannel for flow reaction for 2 h. After the polymerization reaction was completed, 5 mL of 1.0 mol / L CuBr2 solution was injected into the microchannel to make the inner surface of the microchannel a bromide ion end group, completing the block copolymerization.

[0086] (5) 7.00 mmol N-(2-aminoethyl)methacrylamide hydrochloride, 1.40 mmol copper bromide and 2.80 mmol 2,2'-bipyridine were added to 10 mL of a mixed solution of isopropanol and water (isopropanol / water = 8:2 v / v). The mixed solution was stirred with 1.40 mmol [11-(2-bromo-2-methyl)propionyloxy]undecyltrichlorosilane at 50 °C for 6 h.

[0087] (6) After incubating tyrosinase solutions of different concentrations with 25 μg / mL Staphylococcus aureus protein A solution at room temperature for 30 min, the solutions were injected into the capillary microchannel and reacted at room temperature for 1 h.

[0088] (7) Next, 20 μg / mL TCO was dissolved in DMF and injected into the capillary microchannel for incubation at room temperature for 1 h. After the reaction was completed, the microchannel was washed with PBST and blocked with 5% BSA at 37°C for 30 min. After washing with PBST, the microchannel was stored at 4°C until use.

[0089] (8) Dissolve TZ in DMF and stir at room temperature for 1 h at a molar ratio of 20:1 (TZ to biorecognition molecule). After the reaction is completed, add 100 µL Tris-HCl (50 mM) to the mixture to terminate the reaction for 10 min. Add the mixture to a dialysis bag and dialyze at 4 °C for 12 h. Finally, store the prepared TZ-biorecognition molecule conjugate at -20 °C for later use. When testing, dilute to 20 μg / mL and inject into the capillary microchannel. React at room temperature for 15 min to complete the modification process.

[0090] Example 2 Construction of a microchannel current biosensor device based on surface charge changes

[0091] A microchannel current biosensor device based on surface charge change consists of an integrated current sensor device and a millimeter-scale functionalized capillary microchannel. The main component of the capillary microchannel is silicon dioxide, and the click reagent is modified on the inner surface of the microchannel through chemical bonding with the inner wall of the capillary microchannel.

[0092] 1. Construction of an integrated microchannel current biosensor device

[0093] The current sensing device constructed by the present invention integrates the preparation of microchannels and the detection of electrical signals, and is composed of a fully automatic three-axis mobile control element, a pressure system, a reagent tray, electrodes, a power supply, a high-precision online ammeter, and a control and data monitoring panel. The device structure diagram is shown in FIG. Figure 4 shown.

[0094] The fully automatic three-axis mobile control part 2 controls the position of the biochemical reagent, the volume of the solution in the hollow reagent storage bottle 9, and the switch of the power supply of the test system through the touch screen 1; the vacuum pressure system 3 maintains a sealed state with the hollow reagent storage bottle 9 fixed by the rubber stopper 10 to apply pressure to control the injection and removal of the solution; the reagent loading platform 4 is a component for placing biochemical reagents used for chemical modification, biorecognition molecule labeling and sample detection; the two platinum wire electrodes 5 are immersed in the reagent disk 7 and the reagent storage bottle 9 respectively, and form a closed loop with the microchannel 6 fixed by the gasket 8; the microchannel 6 is a solid phase carrier of the biorecognition molecule labeling; the power module provides a power source for the sensing detection device, the fully automatic three-axis mobile control part and the pressure system; the electrical signal monitor transmits the detected electrical signal to the touch screen through the control and data conversion module to realize the collection and processing of the electrical signal. The various operations of the device adjust various parameters through the control panel to realize the automatic function operation.

[0095] 2. Study on the correlation between charge density and current

[0096] Procalcitonin was used as a template molecule, with an inner diameter of 50 μm, a TCO coating concentration of 15 μg / mL, and a capture antibody-TZ concentration of 20 μg / mL. The prepared functionalized capillary microchannel was cut into small segments of 3 mm in length and embedded in the gasket and fixed between two conductive cells. Different concentrations of procalcitonin (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 5000 ng / mL) were injected into the capillary microchannel through a pressure system, and the immune reaction was carried out at room temperature for 20 minutes. After washing with PBST, 20 μg / mL protein spheres containing procalcitonin detection antibodies were added and allowed to react at room temperature for 20 minutes. The microchannel was cleaned with PBST again, and 100 μL of 0.1 mol / L KCl solution was added to each of the two conductive cells. The positive and negative electrodes were inserted and a constant voltage was applied to monitor the change of charge density in the microchannel. A 100 V DC voltage was applied to measure the current in the circuit.

[0097] The results are as follows Figure 5 As shown in the figure, with the increase of procalcitonin concentration, the current shows a clear upward trend. This is because after the capture antibody specifically recognizes the target procalcitonin, the sandwich complex formed by the capture antibody and the protein ball containing the detection antibody increases the protein molecules fixed on the inner surface of the capillary microchannel, thereby increasing the charge density and the current. With the increase of procalcitonin concentration, its response to the current signal is more obvious.

[0098] 3. Optimization of preparation conditions for functionalized capillary microchannels

[0099] The preparation conditions of the functionalized capillary microchannel sensor, such as the inner diameter and length of the microchannel, the applied voltage, the concentration of the click reagent, and the concentration of the coated antibody, were investigated, and the current signal was monitored in real time, as follows:

[0100] Experiment 1) The inner diameter of the microchannel was 25 μm, 50 μm, 75 μm, and 100 μm, respectively, the length was 3 mm, the TCO coating concentration was 15 μg / mL, the TZ-procalcitonin capture antibody concentration was 20 μg / mL, the protein ball concentration containing the procalcitonin detection antibody was 20 μg / mL, and the procalcitonin concentration was 0, 1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 pg / mL, applied voltage was 100 V, and the correlation between the measured current change value (ΔI) and the logarithm of procalcitonin concentration.

[0101] The test results are as follows Figure 6 As shown, the preferred microchannel inner diameter is 50 μm.

[0102] Experiment 2) The inner diameter of the microchannel was 50 μm, and the lengths were 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, and 5 mm, respectively. The TCO coating concentration was 15 μg / mL, the TZ-procalcitonin capture antibody concentration was 20 μg / mL, the protein ball concentration containing the procalcitonin detection antibody was 20 μg / mL, and the procalcitonin concentration was 0, 1, 10, and 10 μg / mL. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 pg / mL, applied voltage was 100 V, and the correlation between the measured current change value (ΔI) and the logarithm of procalcitonin concentration.

[0103] The test results are as follows Figure 7 As shown, the preferred microchannel length is 3 mm.

[0104] Experiment 3) The inner diameter of the microchannel was 50 μm, the length was 3 mm, the TCO coating concentrations were 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL, the TZ-procalcitonin capture antibody concentration was 20 μg / mL, the protein ball concentration containing the procalcitonin detection antibody was 20 μg / mL, the applied voltage was 100 V, and the correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration was obtained.

[0105] The test results are as follows Figure 8 As shown in the figure, the optimal TCO coating concentration is 15 μg / mL.

[0106] Experiment 4) The inner diameter of the microchannel was 50 μm, the length was 3 mm, the TCO coating concentration was 15 μg / mL, the concentrations of TZ-procalcitonin capture antibody were 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL, the concentration of protein spheres containing procalcitonin detection antibody was 20 μg / mL, the applied voltage was 100 V, and the correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration.

[0107] The test results are as follows Fig. 9 As shown, the optimal TZ-procalcitonin capture antibody concentration was 20 μg / mL.

[0108] Experiment 5) The inner diameter of the microchannel was 50 μm, the length was 3 mm, the TCO coating concentration was 15 μg / mL, the TZ-procalcitonin capture antibody concentration was 20 μg / mL, the concentrations of the protein spheres containing the procalcitonin detection antibody were 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL, respectively, and the applied voltage was 100 V. Correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration.

[0109] The test results are as follows Fig.10 As shown, the optimal concentration of protein beads containing procalcitonin detection antibodies is 20 μg / mL.

[0110] Experiment 7) The inner diameter of the microchannel was 50 μm, the length was 3 mm, the TCO coating concentration was 15 μg / mL, the TZ-procalcitonin capture antibody concentration was 20 μg / mL, the protein ball concentration containing the procalcitonin detection antibody was 20 μg / mL, and the applied voltage was 50 V, 100 V, 200 V, and 300 V. The correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration was shown in Figure 7.

[0111] The test results are as follows Fig.11 As shown, the preferred voltage is 100 V.

[0112] Therefore, it was finally determined that the inner diameter of the microchannel was 50 μm, the length was 3 mm, the TCO coating concentration was 15 μg / mL, the capture antibody-TZ concentration was 20 μg / mL, the protein ball concentration containing the procalcitonin detection antibody was 20 μg / mL, and the applied voltage was 100 V for the best detection effect.

[0113] Example 3 Performance evaluation of functionalized capillary microchannels based on directional coupling technology

[0114] 1. Research on the method of directional coupling technology

[0115] The functionalized capillary microchannel sensor prepared by the directional coupling technology based on click reaction has the following steps: the click reagent TCO is injected into the microchannel in advance, and it is allowed to stand at room temperature for 1 h to modify the inner surface with TCO. After washing with PBST, 5% BSA is used at 37°C for 30 min to block the unbound sites, and then it is washed with PBST for use. When it is ready for detection, the procalcitonin capture antibody coupled with TZ is added to induce the click reaction so that the capture antibody is directional coupled and fixed on the inner wall of the microchannel, so as to achieve the purpose of efficient modification and specific capture of the target.

[0116] The conventional non-directional coupling method is: in a carbonate buffer environment, the procalcitonin capture antibody is directly coated on the inner wall of the capillary microchannel at 37℃ for 2h or 4℃ overnight, and 5% BSA is used to block the unbound sites at 37℃ for 30 min. The detection results of the sensors prepared by the two methods for procalcitonin are shown in Table 1. Compared with the non-directional coupling method, the directional coupling method shortens the time by 87.5%, prolongs the storage time by 20 times, and can be completed at room temperature. Among them, the current change is the current change value caused by the same concentration of 1μg / mL procalcitonin. The microchannel sensor with directional coupling technology combined with click reaction has the characteristics of low cost, high sensitivity, fast coupling speed and good stability.

[0117] Table 1

[0118]

[0119] The bioactivity decay degree of the sensors directly coupled to the inner wall of the microchannel with biorecognition molecules and pre-coupled with click reagents was compared. Fig.12 Compared with the traditional method of directly coupling biorecognition molecules, the microchannel sensor using pre-coupled click reagents has stronger adaptability to the environment and better stability.

[0120] For the blocking solution after coupling click reagent 1 in the microchannel, taking the procalcitonin concentration of 1 μg / mL as an example, 1% BSA, 5% BSA, and 10% BSA were selected respectively to measure the current change value (ΔI) under the optimal reaction conditions and the current change value (ΔI) obtained by the blank control when the procalcitonin concentration was 0, and the ratio of specific adsorption to nonspecific adsorption was calculated as shown in Table 2. The specific adsorption ratio of the unbound sites on the inner wall of the microchannel capillary blocked by 5% BSA was the highest, and the effect was the best, so 5% BSA was selected.

[0121] Table 2

[0122]

[0123] 2. Method optimization of protein bead signal amplification system containing detection antibodies

[0124] Taking procalcitonin as an example, the inner wall of the microchannel was modified with 10 μg / mL of procalcitonin capture antibody, the concentration ratios of bovine serum albumin and detection antibody conjugate were 1:1, 4:1, 9:1, and 14:1, respectively, and the applied voltage was 100 V. The correlation between the measured current change value (ΔI) and the logarithm of the procalcitonin concentration was obtained.

[0125] The test results are as follows Fig.13 As shown, the optimal BSA to detection antibody conjugate concentration ratio is 9:1.

[0126] In addition, the signal amplification effect of adding protein balls containing detection antibodies was compared with the current change value caused by only one step of immune reaction. The detection results are as follows Fig.14 After adding the signal amplification system, the same concentration of the analyte can cause a larger current response.

[0127] Example 4 Detection of procalcitonin in serum using a microchannel biosensor method based on surface charge changes

[0128] The functionalized capillary microchannel prepared based on the present invention detects procalcitonin in serum. The principle of detecting procalcitonin is as follows: the procalcitonin capture antibody modified by click reaction in the microchannel reacts immunologically with the procalcitonin in the serum sample, and forms a sandwich complex with the protein balls containing the detection antibody. The greater the procalcitonin concentration in the sample, the more targets are captured on the surface of the functionalized microchannel, the more protein balls are bound, the greater the charge density, and the greater the current response. By comparing the current signal generated by the test sample with the blank current signal, the target content can be accurately analyzed.

[0129] 1. Establishment of standard curve for procalcitonin detection

[0130] First, the type of buffer solution, the time for the immunoreaction between the procalcitonin-coated antibody and procalcitonin, and the time for the protein ball containing the detection antibody to react with procalcitonin were optimized. Fig.15 , Fig.16 , Fig.17 As shown, the optimal buffer solution for the microchannel current biosensor is 0.1 mol / L KCl solution, the optimal immune reaction time is 20 min, and the optimal reaction time of the protein ball containing the detection antibody is 20 min.

[0131] 2. Under the above conditions, establish a standard curve:

[0132] 1) Dissolve 15 μg / mL TCO in DMF, inject into the capillary microchannel and incubate at room temperature for 1 h; after the reaction, wash with PBST, add 5% BSA and block at 37℃ for 30 min, wash with PBST and store at 4℃ for later use;

[0133] 2) Dissolve TZ in DMF, mix and stir at room temperature for 1 h at a molar ratio of TZ: procalcitonin capture antibody of 20:1; after the reaction is completed, add 100 µL Tris-HCl (50 mM) to the above mixture to terminate the reaction for 10 min, add the above mixture to a dialysis bag and dialyze at 4°C for 12 h; finally, store the prepared TZ-procalcitonin capture antibody conjugate at -20°C for use; dilute to 20 μg / mL and inject into the capillary microchannel for detection, and complete the modification process at room temperature for 15 min.

[0134] 3) Procalcitonin standards with concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, and 5000 ng / mL were injected into the capillary microchannel through the fully automatic three-axis motion control element and allowed to react at room temperature for 20 min;

[0135] 4) After washing the microchannel with PBST, add 20 μg / mL procalcitonin detection antibody coupled with protein beads and let react at room temperature for 20 min;

[0136] 5) Clean the microchannel with PBST, add 100 μL of 0.1 mol / L KCl solution to each of the two conductive cells, insert the positive and negative electrodes and apply a constant voltage of 100 V, and monitor the current changes in the circuit in real time through the current detector.

[0137] 6) Draw a standard curve with the logarithmic value of procalcitonin concentration as the horizontal axis and the change value of the current signal as the vertical axis.

[0138] like Fig.18 As shown in Figure 2, the greater the concentration of procalcitonin, the greater the current signal. 6 In the range of pg / mL, there is a good linear relationship between the net chemiluminescence intensity induced by procalcitonin and the logarithmic value of procalcitonin concentration. The regression equation is y=577.11x+1640.33, and the correlation coefficient is R 2 It is 0.989.

[0139] 3. Detection of procalcitonin in serum

[0140] 1) Dissolve 15 μg / mL TCO in DMF, inject into the capillary microchannel and incubate at room temperature for 1 h; after the reaction, wash with PBST, add 5% BSA and block at 37℃ for 30 min, wash with PBST and store at 4℃ for later use;

[0141] 2) Dissolve TZ in DMF, mix and stir at room temperature for 1 h at a molar ratio of TZ: procalcitonin capture antibody of 20:1; after the reaction is completed, add 100 µL Tris-HCl (50 mM) to the above mixture to terminate the reaction for 10 min, add the above mixture to a dialysis bag and dialyze at 4°C for 12 h; finally, store the prepared TZ-procalcitonin capture antibody conjugate at -20°C for use; dilute to 20 μg / mL and inject into the capillary microchannel for 15 min at room temperature to complete the modification process.

[0142] 3) Add 0, 50, 5000, and 500,000 pg of procalcitonin standard to 1 mL of serum sample, inject the sample into the functionalized capillary microchannel through the fully automatic three-axis motion control element, and let it react at room temperature for 20 min;

[0143] 4) After washing the microchannel with PBST, add 20 μg / mL procalcitonin detection antibody coupled with protein beads and let react at room temperature for 20 min;

[0144] 5) Clean the microchannel with PBST, add 100 μL of 0.1 mol / L KCl solution to each of the two conductive cells, insert the positive and negative electrodes and apply a constant voltage of 100 V. Monitor the current in the circuit in real time through the current detector and record the current change value.

[0145] The results are shown in Table 3. The microchannel current biosensor device based on surface charge change was used to monitor procalcitonin in serum. The recoveries were all in the range of 83.86% to 109.30%, and the relative standard deviations were all lower than 10.67%. The comparison results with ELISA detection showed that this method had high stability and accuracy and could be used for the detection of disease markers in serum.

[0146] Table 3. Detection results of procalcitonin in serum samples

[0147]

[0148] Example 5 Detection of chloramphenicol in milk by microchannel immunosensor method based on surface charge change

[0149] The detection principle of chloramphenicol is as follows: the click reagent-chloramphenicol complete antigen is modified into the capillary microchannel by chemical modification. When the target exists in the sample, the chloramphenicol-BSA coupled in the microchannel competes with the target to bind to the capture antibody. When the protein ball containing the detection antibody is added, the higher the concentration of the target, the lower the concentration of the capture antibody bound to the inner surface, the fewer protein balls containing the detection antibody connected to it, the smaller the charge density change, and the smaller the current change value; when there is no target in the sample, more capture antibodies bind to the complete antigen coupled in the microchannel, the more protein balls containing the detection antibody bound to the inner surface, the larger the charge density change, and the increase in the current change value. The chloramphenicol content in the sample can be indirectly obtained by monitoring the change in current.

[0150] 1. Establishment of the standard curve for chloramphenicol detection

[0151] First, the concentration of TZ-chloramphenicol complete antigen and chloramphenicol capture antibody in the modified microchannel were optimized, e.g. Fig.19 The optimal concentration of TZ-chloramphenicol complete antigen in the modified microchannel is 15 μg / mL. Fig. 20 Preferably, the concentration of chloramphenicol capture antibody is 20 μg / mL, and the capillary microchannel fully antigen-functionalized with chloramphenicol is prepared according to the method described in Example 1.

[0152] Next, the time of the competitive reaction for detecting chloramphenicol using the prepared chloramphenicol fully antigen-functionalized capillary microchannel was optimized. Fig.21 As shown, the optimal time for the immunoreaction of the microchannel current biosensor is 15 min.

[0153] 2. Under the above conditions, establish a standard curve:

[0154] The specific process is as follows:

[0155] 1) Dissolve 15 μg / mL TCO in DMF, inject into the capillary microchannel and incubate at room temperature for 1 h; after the reaction, wash with PBST, add 5% BSA and block at 37℃ for 30 min, wash with PBST and store at 4℃ for later use;

[0156] 2) Dissolve TZ in DMF, mix and stir at room temperature for 1 h at a molar ratio of TZ: chloramphenicol complete antigen of 20:1; after the reaction is completed, add 100 µL Tris-HCl (50 mM) to the above mixture to terminate the reaction for 10 min, add the above mixture to a dialysis bag and dialyze at 4°C for 12 h; finally, store the prepared TZ-chloramphenicol complete antigen conjugate at -20°C for use; when testing, dilute the conjugate to 15 μg / mL and inject it into the capillary microchannel, and complete the modification process at room temperature for 15 min.

[0157] 3) Take a certain amount of chloramphenicol standard, prepare a 1 mg / mL chloramphenicol standard stock solution with methanol, and dilute it with PBS to 0, 0.01, 0.1, 1, 10, 100, 1000 ng / mL standard solution;

[0158] 4) Dilute the chloramphenicol capture antibody to 20 μg / mL with PBS, and mix it with chloramphenicol standards of different concentrations in equal volumes; inject 10 μL of the mixed solution into the capillary microchannel through the fully automatic three-axis motion control element, and react at room temperature for 20 min. After the reaction, wash the capillary microchannel with PBST;

[0159] 3) Add 20 μg / mL chloramphenicol detection antibody coupled with protein beads and let react at room temperature for 20 min;

[0160] 4) Wash the microchannel with PBST, add 100 μL 0.1 mol / L KCl electrolyte solution to two sample bottles respectively, and fill the capillary microchannel with electrolyte solution. Insert positive and negative electrodes, apply a constant voltage, and the solution moves toward the negative electrode as a whole. However, the change of surface charge in the capillary microchannel will cause the surface charge density in the microchannel to change. Use a monitor that can monitor the current change in real time to record the current change, and compare it with the blank group to obtain the current difference;

[0161] 5) Draw a standard curve with the logarithm of chloramphenicol concentration as the horizontal axis and the current difference as the vertical axis.

[0162] like Fig. 22 As shown in the figure, there is a good linear relationship between the current difference and the logarithm of chloramphenicol concentration in the range of 0.01~1000 ng / mL, indicating that the method can be used for rapid quantitative detection of chloramphenicol.

[0163] 3. Detection of chloramphenicol content in milk

[0164] The detection process for chloramphenicol in milk is as follows:

[0165] 1) Dissolve 15 μg / mL TCO in DMF, inject into the capillary microchannel and incubate at room temperature for 1 h; after the reaction, wash with PBST, add 5% BSA and block at 37℃ for 30 min, wash with PBST and store at 4℃ for later use;

[0166] 2) Dissolve TZ in DMF, mix and stir at room temperature for 1 h at a molar ratio of TZ: chloramphenicol complete antigen of 20:1; after the reaction is completed, add 100 µL Tris-HCl (50 mM) to the above mixture to terminate the reaction for 10 min, add the above mixture to a dialysis bag and dialyze at 4°C for 12 h; finally, store the prepared TZ-chloramphenicol complete antigen conjugate at -20°C for use; when to be detected, dilute to 15 μg / mL and inject into the capillary microchannel, and complete the modification process at room temperature for 15 min.

[0167] 1) Take 1 mL of milk sample, add 0.05, 5, and 500 ng / mL chloramphenicol standard solution to it, dilute 10 times with PBS, and filter.

[0168] 2) Mix the filtered milk sample with an equal volume of 20 μg / mL chloramphenicol capture antibody, take 10 μL of the mixed solution and inject it into the capillary microchannel through the fully automatic three-axis motion control element, and react at room temperature for 20 minutes. After the reaction, the capillary microchannel is washed with PBST.

[0169] 3) Add 20 μg / mL chloramphenicol detection antibody coupled with protein beads and let react at room temperature for 20 min; after the reaction, wash the capillary microchannel with PBST.

[0170] 4) Add 100 μL of 0.1 mol / L KCl electrolyte solution to each of the two sample bottles, fill the capillary microchannel with electrolyte solution, insert positive and negative electrodes, apply a constant voltage, and test the current change in real time through the current monitor. Compare with the blank group, and quantitatively analyze the target through the current difference. The test results are shown in the following table:

[0171] Table 4. Detection results of chloramphenicol in milk samples

[0172]

[0173] Example 6 Detection of Salmonella in Eggs by Microchannel Biosensing Method Based on Surface Charge Change

[0174] The detection principle of Salmonella is as follows: the click reagent-Salmonella antibody is modified into the capillary microchannel through the click reaction. When Salmonella exists in the sample, the Salmonella antibody coupled in the microchannel will specifically capture the Salmonella. After the protein ball containing the detection antibody is added, the higher the concentration of bacteria, the more live bacteria are fixed on the inner wall surface, and the more protein balls containing the detection antibody are bound. Since the size of bacteria is (0.7-1.5) μm×(2.0-5.0) μm, which is much larger than the size of protein, there is a synergistic effect of size blocking effect and charge change in the detection of the microchannel. The larger the number of Salmonella, the more protein balls containing the detection antibody in the microchannel, the greater the size blocking effect, the smaller the generated current signal, and the greater the signal difference with the blank sample; when there is no target in the sample, fewer Salmonella are captured on the inner surface of the microchannel, the fewer protein balls in the microchannel, the smaller the size blocking effect, the larger the generated current signal, and the smaller the difference with the blank sample. By monitoring the change in current, the content of Salmonella in the sample can be indirectly obtained.

[0175] 1. Establishment of standard curve for detection of Salmonella

[0176] First, the concentration of the Salmonella antibody for the modified microchannel was optimized, e.g. Fig.23 As shown, the optimal concentration of the antibody for modifying the microchannel is 20 μg / mL.

[0177] The time of the immune capture reaction of Salmonella antibody functionalized microchannel was optimized. Fig.24 As shown, the bacterial immune capture reaction time of the microchannel current biosensor is 30 min.

[0178] Under the above conditions, a standard curve was established:

[0179] The specific process is as follows:

[0180] 1) Dissolve 15 μg / mL TCO in DMF, inject into the capillary microchannel and incubate at room temperature for 1 h; after the reaction, wash with PBST, add 5% BSA and block at 37℃ for 30 min, wash with PBST and store at 4℃ for later use;

[0181] 2) Dissolve TZ in DMF, mix and stir at room temperature for 1 h at a molar ratio of TZ:Salmonella capture antibody of 20:1; after the reaction is completed, add 100 µL Tris-HCl (50 mM) to the above mixture to terminate the reaction for 10 min, add the above mixture to a dialysis bag and dialyze at 4°C for 12 h; finally, store the prepared TZ-Salmonella capture antibody conjugate at -20°C for use; dilute to 20 μg / mL and inject into the capillary microchannel for detection, and complete the modification process at room temperature for 15 min.

[0182] 3) Take 1 mL of Salmonella stock solution and add it to TSB solution to multiply to 1×10 7 CFU / mL, and diluted with PBS to 0, 10, 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 CFU / mL;

[0183] 4) Take 10 μL of diluted bacterial solution and place it in different sample areas of the reagent disk. Use the fully automatic three-axis motion control element to inject the bacterial solution into the microchannel of the tube and react at 37°C for 30 min. After the reaction, wash the capillary microchannel with PBST;

[0184] 5) Add 20 μg / mL of Salmonella detection antibody coupled with protein beads and let it react at room temperature for 20 min. After the reaction, wash the capillary microchannel with PBST;

[0185] 6) Add 100 μL of 0.1 mol / L KCl electrolyte solution to each of the two sample bottles, fill the capillary microchannel with the electrolyte solution, insert the positive and negative electrodes, apply a constant voltage, and the solution moves toward the negative electrode as a whole. However, the change in the surface charge of the capillary microchannel will cause the surface charge density of the microchannel to change. Use a monitor that can monitor the current change in real time to record the current change, and compare it with the blank group to obtain the current difference;

[0186] 7) Draw a standard curve with the logarithm of Salmonella concentration as the horizontal axis and the current difference as the vertical axis.

[0187] like Fig.25 As shown, in 1×10 2 ~1×10 6 There is a good linear relationship between the current difference and the logarithm of the Salmonella concentration in the CFU / mL range, indicating that the method can be used for rapid quantitative detection of Salmonella.

[0188] 3. Detection of Salmonella content in eggs

[0189] The detection process for Salmonella in eggs is as follows:

[0190] 1) Take 1 mL of egg sample and add 1 mL of standard solution with a concentration of 1×10 2 , 1×10 4 , 1×10 6 CFU of Salmonella solution was cultured in 4 mL TSB liquid medium for 6 h;

[0191] 2) Dilute the cultured solution 10 times with PBS and filter;

[0192] 3) Take 10 μL of the mixed solution and place it in different sample areas of the reagent disk and inject it into the microchannel through the fully automatic three-axis motion control element, and react at room temperature for 30 minutes. After the reaction, the capillary microchannel is washed with PBST;

[0193] 4) Add 20 μg / mL of Salmonella detection antibody coupled with protein beads and let it react at room temperature for 20 min. After the reaction, wash the capillary microchannel with PBST; After the reaction, wash the capillary microchannel with PBST;

[0194] 5) Add 100 μL of 0.1 mol / L KCl electrolyte solution to each of the two sample bottles, fill the capillary microchannel with electrolyte solution, insert positive and negative electrodes, apply a constant voltage, test the current change in real time through the current monitor, and compare with the blank group. The target is quantitatively analyzed by the current difference. The test results are shown in the following table:

[0195] Table 5. Results of detection of Salmonella in egg samples

[0196]

[0197] The above analysis method can also be achieved by continuously monitoring voltage changes. We pre-modify the click reagent in the microchannel reaction area, add the biorecognition molecule coupled with the click reagent when testing, induce the click reaction to achieve the coating process, and then add the target to be tested for incubation. After the biorecognition molecule specifically captures the target substance, add the protein ball containing the detection antibody. The protein ball will cause the surface charge density in the microchannel to change. A constant current is applied at both ends of the channel, and different voltages will be generated at both ends of the microchannel. By detecting the change in voltage, the target substance can be quantitatively analyzed.

Claims

1. A charge-mediated microchannel current biosensing method based on directional coupling technology, characterized in that: The method comprises the following steps: S1 pre-modifies the click reagent 1 on the inner surface of the microchannel, and when testing, adds the biorecognition molecule coupled with the click reagent 2, induces the click reaction so that the biorecognition molecule is directionally coupled and fixed on the inner wall of the microchannel, and sequentially adds the target to be tested and the self-assembled protein sphere containing the detection antibody and bovine serum albumin to perform the immune capture reaction. The protein sphere can amplify the signal of each antigen-antibody specific binding reaction, so that the charge state of the inner surface of the microchannel changes, and the change in charge density is related to the concentration of the target to be tested; The S2-modified microchannel is fixed in the middle of the two conductive liquid pools, becoming the only channel connecting the two conductive liquid pools. The positive and negative electrodes are respectively inserted into the conductive cells at both ends of the microchannel and a constant voltage or current is applied, forming a closed-loop circuit between the electrode-electrolyte-microchannel. The S3 sample solution passes through the microchannel, undergoing a specific biorecognition reaction, and then self-assembled protein balls containing detection antibodies and bovine serum albumin are added. Since the protein surface carries a negative charge, the aggregation of proteins into protein balls can cause a greater change in the charge density in the microchannel, which in turn causes a change in the current of the closed-loop circuit. The degree of change is related to the charge density in the microchannel, so that the content of the target substance to be measured can be obtained; In the step S1, click reagent 1 and click reagent 2 include tetrazine group (TZ)-trans-cyclooctene (TCO), azide-alkyne, dibenzocyclooctyne (DBCO)-azide (Azide), cysteine ​​(Cys)-cyanobenzothiazole (CBT) or others; The biorecognition molecule is an antibody or antigen that can react with the target to be detected, or the biorecognition molecule is a bacteriophage that can specifically capture microorganisms, or the biorecognition molecule is a nucleic acid aptamer of a capture probe that can react with the target to be detected by DNA molecule hybridization, or the biorecognition molecule is a nanobody that specifically captures the target to be detected; The concentration of the biorecognition molecules modifying the inner wall of the microchannel is 1 μg / mL to 30 μg / mL; when a constant voltage is applied to the positive electrode and the negative electrode, the voltage is 50-300V.

2. The charge-mediated microchannel current biosensing method based on directional coupling technology according to claim 1, characterized in that: In the step S1, the microchannel is a capillary, and the biorecognition molecules are modified on the inner surface of the microchannel by directional coupling technology, and the specific operation includes the following steps: S1 A.1 Rinse the capillary with hydrochloric acid to remove contaminants attached to the inner wall; S1 A.2 Continue to rinse the capillary with sodium hydroxide to activate the silanol groups on the inner wall of the capillary; S1 A.3 Take the surface-immobilized atom transfer radical polymerization initiator [11-(2-bromo-2-methyl)propionyloxy] undecyltrichlorosilane (5 wt%, dissolved in toluene) and inject it into the capillary microchannel for reaction. After the reaction is completed, the microchannel is cleaned with toluene and methanol in turn; S1A.4 Silicon-based block graft copolymerization: 2-methacryloyloxyethyl phosphorylcholine, copper bromide and 2,2'-bipyridine are added to degassed methanol, and the mixed solution is injected into the capillary microchannel for polymerization reaction; After the polymerization reaction is completed, a CuBr2 solution is injected into the microchannel to make the inner surface of the microchannel a bromide ion end group, thus completing the block copolymerization; S1A.5 N-(2-aminoethyl)methacrylamide hydrochloride, copper bromide and 2,2'-bipyridine are added to a mixed solution of isopropanol and water (isopropanol / water = 8:2 v / v), and the mixed solution is stirred with [11-(2-bromo-2-methyl)propionyloxy]undecyltrichlorosilane; S1A.6 After incubating the tyrosinase solution and the Staphylococcus aureus protein A solution at room temperature, the solution was injected into the capillary microchannel and reacted at room temperature; S1A.7 Dissolve TCO in DMF, inject into the capillary microchannel and incubate at room temperature; after the reaction, wash with PBST, add 5% BSA to block, wash with PBST and store at 4°C for future use; S1 A.8 Take TZ and dissolve it in DMF, mix TZ and the biorecognition molecule at room temperature; after the reaction is completed, add Tris-HCl to the above mixture to terminate the reaction, and add the above mixture to a dialysis bag for dialyzing; finally, store the prepared TZ-biorecognition molecule conjugate at -20℃ for future use; when it is to be detected, inject it into the capillary microchannel to complete the modification process.

3. The charge-mediated microchannel current biosensing method based on directional coupling technology according to claim 1, characterized in that: The self-assembled protein spheres in step S1 include detection antibodies and bovine serum albumin, horseradish peroxidase, alkaline phosphatase or others; the protein sphere self-assembly process includes the following steps: S1B.1 Mix bovine serum albumin solution, detection antibody and surfactant CTAB in phosphate buffer, and the three will synthesize protein nanoconjugates through electrostatic reaction; S1B.2 isooctyl alcohol is added to the aqueous solution of the nanoconjugate to form a protein ball dispersion, and then glutaraldehyde is added to react the exposed amine with glutaraldehyde to cross-link the protein molecules in the surface membrane of the protein ball and react at the water / oil interface; S1B.3 The protein spheres are dialyzed against a water / ethanol mixture to remove isooctyl alcohol and CTAB to ensure that the protein spheres are transferred to the aqueous phase without membrane collapse.

4. Application of the charge-mediated microchannel current biosensing method based on directional coupling technology as described in any one of claims 1 to 3 for detecting antibiotics, pathogenic microorganisms or agricultural and veterinary drugs for non-diagnostic purposes.

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