A method for detecting pathogenic bacteria based on Fe-N-C nanozyme signal amplification colorimetric biosensor and microfluidic chip

Abstract

The application provides a colorimetric biosensor and a microfluidic chip based on Fe-N-C nanoscale enzyme signal amplification for detecting pathogenic bacteria. The Fe-ZIF8 precursor is synthesized by a one-pot method, and after high-temperature calcination, the Fe-N-C nanoscale enzyme with high simulated enzyme activity is obtained. The nanoscale enzyme is used to replace the traditional biological enzyme (such as HRP) to label the target bacteria, so that the color change of the target bacteria is more obvious, and the parameters are optimized to match the linear detection range of the smart phone App. Finally, a gravity microfluidic chip is developed to perform the bacterial detection step, and the fluid flow is further optimized by optimizing the channel size. The application provides a powerful tool for the detection of foodborne pathogenic bacteria.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemical analysis, in particular to a method for detecting pathogenic bacteria based on Fe-N-C nanoscale enzyme signal amplification colorimetric biosensor and microfluidic chip. BACKGROUND

[0002] Metal-organic frameworks (MOFs) are porous materials that are self-assembled from inorganic metals as centers and organic ligands. They have attracted much attention due to their highly ordered porosity, tunable pore size, and atomically dispersed metal nodes. The dual-metal MOF materials doped with transition metals not only have enzyme activity, but also have highly ordered porosity and atomically dispersed metal nodes, thus forming a unique confinement effect and catalytic microenvironment, which enables the substrate to interact with the active site and improves the catalytic performance. Therefore, dual-metal MOF materials are considered as ideal simulated enzymes. However, the stability of dual-metal MOF materials is poor, and many of their properties are easily reduced by external environmental stimuli, such as crystallinity and porosity. In combination with its possible failure mechanism, it is necessary to explore new methods to promote the development of MOF-derived materials to have higher stability. Zeolitic imidazolate framework (ZIF) materials have high mechanical stability, and doping transition metal elements and pyrolysis at high temperature not only can maintain the overall crystallinity and morphology of ZIF materials, but also can fully expose the active sites of the metal loaded by the porous carbon carrier, greatly improving the catalytic ability. Interestingly, the reactivity and instability of the coordination bond in MOF make it easy to be converted into other forms in the pyrolysis reaction, thereby affecting its physical and chemical properties. After pyrolysis of ZIF-8, more Fe catalytic properties are exhibited to obtain Fe-N-C nanoscale enzymes with simulated enzyme activity.

[0003] Gravity microfluidic chip is a kind of micro-laboratory device that combines microfluidic technology and gravity control, which can be used for biological, chemical and physical experiments. It can control and operate small volume of liquid samples for mixing, separation, transportation and detection of liquid samples. Gravity microfluidic chip usually has low manufacturing cost. This makes them more easily adopted, especially in resource-limited laboratory environments, and standard microfabrication techniques such as photolithography, PDMS (polydimethylsiloxane) microfabrication and 3D printing are usually used to manufacture gravity microfluidic chips, which have been widely developed and the manufacturing process is relatively easy to master. And it can usually achieve highly sensitive experiments that can detect and analyze small concentrations of compounds or biological molecules. Therefore, combining it with colorimetric biosensor can greatly improve its sensitivity. SUMMARY

[0004] The purpose of the present application is to provide a method for detecting pathogenic bacteria based on Fe-N-C nanoscale enzyme signal amplification colorimetric biosensor and microfluidic chip.

[0005] To achieve the object of the present application, in a first aspect, the present application provides a method for constructing a colorimetric biosensor based on Fe-N-C nanoscale enzyme signal amplification, comprising the following steps:

[0006] (1) Preparation of Fe-N-C nanoscale enzyme.

[0007] (2) Preparation of aminated Fe-N-C nanoscale enzyme.

[0008] (3) Preparation of immunonanoparticle, i.e. aminated Fe-N-C nanoscale enzyme coupled with first antibody.

[0009] (4) Preparation of immunomagnetic beads, i.e. magnetic beads coupled with second antibody.

[0010] Further, the preparation method of the Fe-N-C nanoscale enzyme in step (1) comprises:

[0011] a. Mixing Zn(NO3)2·6H2O, Fe(NO3)3·9H2O and methanol to obtain solution A;

[0012] b. Mixing 2-methylimidazole and methanol to obtain solution B;

[0013] c. Mixing solution A and solution B to obtain Fe-ZIF-8 precursor; and finally pyrolyzing at 800-900°C under N2 to obtain Fe-N-C nanoscale enzyme.

[0014] More preferably, in one of the more specific embodiments, the preparation method of the Fe-N-C nanoscale enzyme in step (1) comprises:

[0015] a. Mixing a mixture of 2.185 g of Zn(NO3)2·6H2O and 0.065 g of Fe(NO3)3·9H2O with 100 mL of methanol to obtain solution A.

[0016] b. Mixing 2-methylimidazole with 100 mL of methanol to obtain solution B.

[0017] c. Mixing solution A and solution B and stirring at room temperature to obtain Fe-ZIF-8 precursor; washing the Fe-ZIF-8 precursor several times, freeze-drying to obtain solid powder, and finally pyrolyzing at 800-900°C under N2 for 1.5-2h to obtain Fe-N-C nanoscale enzyme.

[0018] Wherein, the molar ratio of Zn(NO3)2·6H2O and Fe(NO3)3·9H2O is (11-15):1, and the molar ratio of 2-methylimidazole to zinc ion is (10-12):1.

[0019] The first antibody of step (3) and the second antibody of step (4) are different antibodies against the same pathogenic bacteria, and the first antibody and the second antibody respectively bind to different epitopes of the pathogenic bacteria.

[0020] Further, the preparation method of the amino-functionalized Fe-N-C nanoszyme of step (2) comprises:

[0021] The Fe-N-C nanoszyme is dissolved in anhydrous ethanol, APTES is added, the pH is adjusted to 10.5-11.0 with ammonia water, and then stirring is performed in a water bath at 50-65°C for 5-8h to obtain the amino-functionalized Fe-N-C nanoszyme.

[0022] More preferably, in one of the more specific embodiments, the preparation method of the amino-functionalized Fe-N-C nanoszyme of step (2) comprises:

[0023] 50 mg of Fe-N-C nanoszyme is weighed and dissolved in 100 mL of anhydrous ethanol, after ultrasonic dispersion, 6-10 mL of APTES is added, the pH is adjusted to 10.5-11.0 with ammonia water, and then stirring is performed in a water bath at 50-65°C for 5-8h, and then the residual APTES is washed with anhydrous ethanol to prepare a functional nanoszyme with conjugated amino groups on the surface, freeze-dried to obtain the amino-functionalized Fe-N-C nanoszyme.

[0024] The pathogenic bacteria include but are not limited to Salmonella ( Salmonella ), such as Salmonella typhimurium ( Salmonella typhimurium ).

[0025] When the pathogenic bacteria are Salmonella, the first antibody is Salmonella monoclonal antibody, and the second antibody is Salmonella polyclonal antibody.

[0026] Further, step (3) comprises: mixing the Salmonella monoclonal antibody with the amino-functionalized Fe-N-C nanoszyme, adding 5%-10% BSA for blocking for 30 min, and finally reconstituting with 1%-3% BSA to obtain the immunonanoszyme.

[0027] More preferably, in one of the more specific embodiments, step (3) comprises: taking 10-30 μg of Salmonella monoclonal antibody and adding it to 1 mL of amino-functionalized Fe-N-C nanoszyme, magnetically stirring, electrostatically adsorbing for 2-5 h, then adding 100 μL of 5%-10% BSA prepared with ultrapure water for blocking for 30 min, washing several times (such as 3 times) with ultrapure water, and finally reconstituting with 1 mL of 1%-3% BSA to obtain the immunonanoszyme, which is placed at 4°C for standby.

[0028] Further, the step (4) comprises: adding the EDC and NHSS mixed solution to the magnetic beads, activating for 1 h to obtain the immunomagnetic beads; after recovering the magnetic beads by magnetic separation, adding the salmonella polyclonal antibody, coupling for 2-4 h; adding the blocking solution to the coupling product for blocking, recovering the magnetic beads by magnetic separation, and resuspending with the resuspension solution.

[0029] More preferably, in one more specific embodiment, the step (4) comprises:

[0030] 1) Magnetic bead washing: 0.8-1 mL PB buffer is first added to a 1.5 mL centrifuge tube, then 0.2 mL of 10 mg / mL magnetic beads are added, and 1 mL of PB buffer is used to wash several times, and then resuspended in 1-2 mL of PB buffer (ultrasonic before each washing to keep the magnetic beads suspended).

[0031] 2) Activation of carboxyl group: the EDC and NHSS mixed solution is added to the magnetic beads with a particle size of 180 nm, and after uniform mixing, the magnetic beads are kept suspended and slowly rotated on the homogenizer, and activated at room temperature for 1 h to obtain the immunomagnetic beads; after the reaction is completed, the state of the magnetic beads is observed to ensure that the state is good and there is no aggregation phenomenon.

[0032] The preparation method of the EDC and NHSS mixed solution comprises: dissolving EDC 0.58 mg and NHSS 0.65 mg in 0.01M pH6.0 PB solution to obtain a mixed solution with a concentration of 1 mg / mL.

[0033] 3) Coupling of salmonella polyclonal antibody: after recovering the magnetic beads by magnetic separation, 1 mL of PB buffer is used to wash several times, and resuspended in 1 mL of PB buffer, 15-25 μg of salmonella polyclonal antibody is added, and placed on the homogenizer, and coupled at 37°C for 2-4 h.

[0034] 4) Blocking: 1% BSA is added to the coupling product of 3) at room temperature for 45-70 min. The magnetic beads are recovered by magnetic separation, washed with 1 mL of PB buffer several times, and resuspended with 1 mL of resuspension solution.

[0035] The preparation method of the resuspension solution is as follows: 2.5 g of sucrose, 100 mg of skim milk and 70 μL of Proclin300 are dissolved in 10 mL of PBS solution with a concentration of 0.01M pH7.4 to obtain the resuspension solution.

[0036] The concentration of the PB buffer is 0.01M, and the pH is 7.4.

[0037] In a second aspect, the present application provides a microfluidic chip for colorimetric biosensor based on Fe-N-C nanoscale enzyme signal amplification, which is a gravity-driven microfluidic chip, comprising a sample inlet, an air hole, a plurality of flow channels, and the following six chambers: a reaction chamber, a washing liquid chamber, a TMB-H2O2 system chamber, a separation chamber, a waste liquid chamber, and a detection chamber; wherein the reaction chamber is pre-embedded with a weak magnetic small iron ball, and the capture rate is improved by mixing the sample, the immunomagnetic beads, and the immunonanoplasma with the weak magnetic small iron ball; wherein the immunomagnetic beads and the immunonanoplasma are the same as the immunomagnetic beads and the immunonanoplasma in the method of any one of claims 1-5.

[0038] More preferably, in a more specific embodiment, the microfluidic chip comprises a sample inlet, an air hole, a plurality of flow channels (flow channels), and the following six chambers: a reaction chamber, a washing liquid chamber, a TMB-H2O2 system chamber, a separation chamber, a waste liquid chamber, and a detection chamber.

[0039] As shown in Figure 1 The size of the microfluidic chip is 8.6 cm in length, 6.8 cm in width, and 0.55 cm in thickness.

[0040] The material of the microfluidic chip is PDMS, and each structural unit is formed by pouring; that is, the microfluidic chip is composed of two layers of PDMS layers with equal thickness, which are engraved with flow channels and chambers.

[0041] All chambers are subjected to surface hydrophobization treatment; all chambers are provided with an air hole for balancing air pressure, and the position of the air hole is higher than the liquid level in the chamber to ensure that the reagents in the chamber do not leak.

[0042] The microfluidic chip mainly comprises: a reaction chamber with a volume of 300 µL, a washing liquid chamber with a volume of 300 µL, a TMB-H2O2 system chamber with a volume of 100 µL, a separation chamber with a volume of 400 µL, a waste liquid chamber with a volume of 800 µL, and a detection chamber with a volume of 200 µL, and a plurality of flow channels with a diameter of 3 mm, and the reaction chamber, the washing liquid chamber, and the TMB-H2O2 system chamber are respectively communicated with the separation chamber through the flow channels, and the detection chamber and the waste liquid chamber are respectively communicated with the separation chamber through the flow channels.

[0043] The reaction chamber is pre-embedded with a weak magnetic small iron ball with a diameter of 3 mm, and the capture rate is improved by mixing the sample, the immunomagnetic beads, and the immunonanoplasma with the weak magnetic small iron ball; wherein the immunomagnetic beads and the immunonanoplasma are the same as the immunomagnetic beads and the immunonanoplasma in the method of any one of claims 1-5.

[0044] The separation chamber is pre-embedded with a neodymium iron boron ball (neodymium iron boron magnetic ball) with a diameter of 3 mm, which is used to capture magnetic beads (magnetic separation is realized based on the magnetism of the neodymium iron boron ball).

[0045] The TMB-H2O2 system chamber is used to store the TMB-H2O2 system.

[0046] The cleaning liquid chamber is used to store the cleaning liquid.

[0047] The waste liquid chamber is used to collect the waste liquid.

[0048] The TMB-H2O2 system is released from the TMB-H2O2 system chamber into the separation chamber for color development reaction, and after the reaction is completed, it is released into the detection chamber, and the product image is collected and analyzed by using a smartphone image analysis App to realize the detection of pathogenic bacteria in the sample.

[0049] Since the pouring material has a certain hydrophobicity, in order to make the fluid in the flow channel flow smoothly, the size of the flow channel is optimized, and the size of the optimized flow channel is 3 mm.

[0050] In a third aspect, the application provides the use of the microfluidic chip in the qualitative and quantitative detection of pathogenic bacteria in a sample (including non-disease diagnosis purposes).

[0051] In a fourth aspect, the application provides a method for detecting pathogenic bacteria based on Fe-N-C nanozyme signal amplification colorimetric biosensor and microfluidic chip (including non-disease diagnosis purposes), which uses the microfluidic chip to detect pathogenic bacteria in a sample.

[0052] The method comprises the following steps:

[0053] The immunomagnetic beads (immunonanomagnetic beads MNBs), the sample solution and the immunonanozyme (aminoated Fe-N-C nanozyme) are pre-mixed and injected into the reaction chamber of the microfluidic chip, and after a mixing reaction for a period of time using an external magnet to stir the weak magnetic small iron ball, it is released to the separation chamber through the flow channel by adjusting and controlling the air hole switch, the double-antibody sandwich complex is captured by the built-in neodymium iron boron ball, and after the supernatant is released to the waste liquid chamber, the washing liquid in the washing liquid chamber is released, the non-specific adsorption on the surface of the magnetic beads is washed, and the supernatant is released to the waste liquid chamber; then, the TMB-H2O2 system in the TMB-H2O2 system chamber is released to the separation chamber, and the color developing substrate is catalyzed by the immunonanozyme on the double-antibody sandwich complex; after the catalytic reaction is completed, it is released to the detection chamber, and the catalytic product is analyzed by using a smartphone image analysis App in the detection chamber to determine the concentration of pathogenic bacteria in the sample.

[0054] By means of the technical scheme, the application has at least the following advantages and beneficial effects:

[0055] The application synthesizes Fe-ZIF8 precursor by one-pot method, and obtains Fe-N-C nanoscale enzyme with high simulated enzyme activity after high-temperature calcination. The nanoscale enzyme is used to replace traditional biological enzyme (such as HRP) to mark target bacteria, so that the color change of the target bacteria is more obvious. The parameters are optimized to match the linear detection range of the smartphone App. Finally, a gravity microfluidic chip is developed to perform the bacterial detection step, and the fluid flow is further optimized by optimizing the channel size. The reaction principle is as follows: the sensor uses salmonella typhi antibody as a recognition element to detect salmonella in food. Fe-ZIF-8 precursor is prepared by adding Fe in the synthesis system, and Fe-N-C nanoscale enzyme is prepared by high-temperature calcination. Amino modification is performed on the Fe-N-C nanoscale enzyme by using (3-aminopropyl) triethoxysilane (APTES). Salmonella typhi monoclonal antibody is coupled with the amino-functionalized Fe-N-C nanoscale enzyme, and salmonella typhi polyclonal antibody is coupled with carboxylated magnetic beads. When the target exists, the target, the Fe-N-C nanoscale enzyme and the immunomagnetic beads form a sandwich structure. After magnetic separation, the Fe-N-C nanoscale enzyme combined with the target is transferred to the precipitate. The precipitate has good peroxidase activity and can catalyze the TMB-H2O2 system, and the color change degree is positively correlated with the concentration of salmonella. The method has high sensitivity, repeatability and specificity. BRIEF DESCRIPTION OF DRAWINGS

[0056] In order to more clearly illustrate the technical solutions in the application or prior art, the following will describe the drawings needed to be used in the embodiments or prior art description.

[0057] Figure 1 It is a structural schematic diagram of the gravity microfluidic chip of the application. Among them, 1 is a reaction chamber, 2 is a washing liquid chamber, 3 is a TMB-H2O2 system chamber, 4 is a detection chamber, 5 is a waste liquid chamber, and 6 is a separation chamber.

[0058] Figure 2 It is a study on the TMB-H2O2 catalytic performance of the Fe-N-C nanoscale enzyme prepared by the application.

[0059] Figure 3 It is a schematic diagram of the principle of the colorimetric biosensor combined with the gravity microfluidic chip in the preferred embodiment of the application.

[0060] Figure 4 It is an experimental result of different concentrations of Fe-N-C catalyzing TMB-H2O2 in the preferred embodiment of the application.

[0061] Figure 5Optimization results of the amount of immunomagnetic beads in the preferred embodiment of the present application.

[0062] Figure 6 Optimization results of the mixing frequency and incubation time in the preferred embodiment of the present application.

[0063] Figure 7 Optimization results of the amount of immunomagnetic Fe-N-C nanoscale enzyme in the preferred embodiment of the present application.

[0064] Figure 8 Optimization results of the catalytic time in the preferred embodiment of the present application.

[0065] Figure 9 Standard curve of the sensor in the preferred embodiment of the present application.

[0066] Figure 10 Performance evaluation of the sensor in the preferred embodiment of the present application: (A) specificity; (B) repeatability. DETAILED DESCRIPTION

[0067] The present application aims to provide a colorimetric biosensor based on a gravity microfluidic chip and Fe-N-C nanoscale enzyme signal amplification, which can be used for rapid detection of foodborne pathogenic bacteria Salmonella typhimurium.

[0068] The present application adopts the following technical solutions:

[0069] Ordinary microfluidic chips usually need external electronic devices or high-pressure systems to provide driving force, such as electric field or pressure. This increases the complexity and cost of the device, and requires additional energy support, which has high preparation and maintenance costs. This makes it less practical in resource-limited situations, especially for some small laboratories or individual users. Gravity microfluidic chips use gravity as a driving force to drive fluids, without the need for complex external equipment or circuit control, and the operation is relatively simple. This reduces the complexity and use threshold of the device, making it easier for non-professionals to use. Gravity microfluidic chips can achieve micro-volume liquid manipulation and mixing, and can quickly and efficiently perform chemical analysis combined with colorimetry. This combination can improve the speed and efficiency of analysis, save reagent and sample consumption, and reduce experimental operation time. In order to improve the sensitivity and stability of the microfluidic chip, nanomaterial modification and nanostructure technology are also used to further enhance the sensitivity and stability of the detection of Salmonella typhimurium.

[0070] The specific process is as follows: (1) synthesis of Fe-N-C nanoscale enzyme; (2) amination of Fe-N-C nanoscale enzyme; (3) nanoscale enzyme coupling monoclonal antibody; (4) magnetic bead coupling polyclonal antibody; (5) design of gravity microfluidic chip suitable for function; (6) optimization of experimental conditions; (7) determination of Salmonella typhimurium.

[0071] Preferably, (1) the preparation method of Fe-N-C nanoszyme is: first, all glassware in the preparation process is immersed in freshly prepared aqua regia for 12 h, and then washed with ultrapure water for cleaning; then, iron ions are added in the MOF material synthesis system to prepare Fe-ZIF-8 precursor. A mixture of 2.185 g Zn(NO3)2·6H2O and 0.065 g Fe(NO3)3·9H2O with a molar ratio of (11-15):1 is mixed with 100 mL methanol to obtain solution A; at the same time, 2-methylimidazole is mixed with 100 mL methanol (the molar ratio of 2-methylimidazole to zinc ions is (10-12):1) to obtain solution B, and then the mixture of solution A and solution B is stirred at room temperature. The Fe-ZIF-8 precursor is washed several times, freeze-dried to obtain a solid powder, and finally pyrolyzed at 800-900°C for 1.5-2 hours under N2 using a tube furnace.

[0072] The Fe-N-C nanoszyme prepared according to the above method has catalytic performance of catalyzing TMB-H2O2 Figure 2 ).

[0073] Preferably, the amino-functionalized Fe-N-C nanoszyme in (2) has the following specific steps: 50 mg of Fe-N-C nanoszyme is weighed and dissolved in 100 mL of anhydrous ethanol. After ultrasonic dispersion, 6-10 mL of APTES is added, the pH is adjusted to 10.5-11.0 with ammonia water, and then stirred in a 55-65°C water bath for 5-8 h. Then, the uncoupled APTES is washed with anhydrous ethanol to prepare a functional nanoszyme with conjugated amino groups on the surface. The amino-functionalized nanoparticles are freeze-dried and stored at 4°C.

[0074] Preferably, the nanoszyme conjugated monoclonal antibody in (3) has the following specific steps: 10-30 μg of Salmonella monoclonal antibody is added to 1 mL of amino-functionalized Fe-N-C nanoszyme, magnetically stirred, and electrostatically adsorbed for 2-5 h. Then, 100 μL of 5-10% BSA prepared with ultrapure water is slowly added and blocked for 30 min. The solution is washed several times with ultrapure water, and finally resuspended in 1 mL of 1% BSA and stored at 4°C.

[0075] Preferably, the magnetic bead-conjugated polyclonal antibody in (4) is prepared by the following method:

[0076] 1) Magnetic bead washing: 0.8-1.0 mL of PB buffer is added to a 1.5 mL centrifuge tube, 0.2 mL of 10 mg / mL magnetic beads is added, and 1 mL of PB buffer is used to wash several times, and then resuspended in 1 mL of PB buffer (ultrasonic before each washing to keep the magnetic beads in suspension);

[0077] 2) Activation of carboxyl group: EDC and NHSS mixed solution was added to magnetic beads with a particle size of 180 nm, and after uniform mixing, the magnetic beads were kept suspended and slowly rotated on a mixing instrument, activated at room temperature for 1 h, and immune magnetic beads were obtained; after the reaction was completed, the state of the magnetic beads was observed to ensure that the state was good and there was no aggregation phenomenon;

[0078] The preparation method of the EDC and NHSS mixed solution comprises: dissolving EDC 0.58 mg and NHSS 0.65 mg in 0.01M pH6.0 PB solution to obtain a mixed solution with a concentration of 1 mg / mL;

[0079] 3) Coupling of polyclonal antibody: after the magnetic beads were recovered by magnetic separation, they were washed several times with 1 mL of PB buffer, resuspended in 1 mL of PB buffer, 15-25 μg of polyclonal antibody was added, and placed in a mixing instrument, coupled at 37°C for 2-4 h;

[0080] 4) Blocking: the coupling product was added to a final concentration of 1% BSA, and blocked at room temperature for 45 min. The magnetic beads were recovered by magnetic separation, washed several times with 1 mL of PB buffer (0.01M, pH 7.4), and resuspended with 1 mL of resuspension solution.

[0081] The preparation method of the resuspension solution is as follows: 2.5 g of sucrose, 100 mg of skim milk and 70 μL of Proclin 300 were dissolved in 10 mL of PBS solution with a concentration of 0.01M pH7.4, and the resuspension solution was obtained.

[0082] Preferably, (5) the gravity microfluidic chip is designed to be suitable for the function; the microfluidic chip is designed to be 8.6 cm long, 6.8 cm wide, and 0.55 cm thick, mainly containing a sample injection hole, an air hole, a chamber, and small iron balls of different materials; (1) there are 6 chambers, and immune magnetic beads (immune nanometer magnetic beads MNBs), sample solution, and immune nanometer enzyme (amino-Fe-N-C nanometer enzyme) are pre-mixed and injected into the reaction chamber of the microfluidic chip at the same time, the weak magnetic small iron balls are stirred and mixed for a period of time using an external magnet, and then the mixture is released into the separation chamber through the air hole switch adjustment control, the double-antibody sandwich complex formed in the separation chamber is captured to the surface by the built-in neodymium iron boron ball, the supernatant is released into the waste liquid chamber after the air hole is opened, the washing liquid in the washing liquid chamber is released, the non-specific adsorption on the surface of the magnetic beads is washed, and the supernatant is released into the waste liquid chamber; then, the TMB-H2O2 system in the TMB-H2O2 system chamber is released into the separation chamber, and the color developing substrate is catalyzed by the immune nanometer enzyme on the double-antibody sandwich complex (i.e., the TMB-H2O2 system is released into the separation chamber to combine with the Salmonella-magnetic bead-nanometer enzyme complex adsorbed on the neodymium iron boron ball, and the reaction lasts for about 15 min). After the catalytic reaction is completed, it is released into the detection chamber, and the catalytic product is analyzed in the detection chamber using a smartphone image analysis App to determine the concentration of pathogenic bacteria in the sample.

[0083] Preferably, (6) optimization of experimental conditions, in order to achieve excellent experimental results, the capture efficiency is optimized by optimizing the mixing frequency of small iron balls, the amount of magnetic beads, and the amount of antibody

[0084] Preferably, (7) determination of Salmonella typhimurium, colorimetric biosensor based on Fe-N-C nanometer enzyme and microfluidic chip: a new type of colorimetric biosensor is constructed by using Fe-N-C nanometer enzyme as horseradish peroxidase (HRP) simulation enzyme, which realizes the simple and rapid detection of Salmonella typhimurium. First, the immune magnetic beads, bacterial samples, and immune Fe-N-C nanometer enzyme are injected into the microfluidic chip at the same time, and the weak magnetic small iron balls are used for mixing and incubation to form a magnetic bead-bacterium-nanometer flower double-antibody sandwich complex, and the complex is captured in the separation chamber by the pre-embedded neodymium iron boron ball to realize the separation and enrichment of the target bacteria; then, single-component tetramethylbenzidine (TMB) is used as a color developing substrate, and the Fe-N-C nanometer enzyme on the complex catalyzes the color change of the substrate, finally, the catalytic product is transferred to the detection chamber, and the image of the product is collected and analyzed by a smartphone image analysis App to realize the quantitative detection of Salmonella typhimurium. The biosensor innovatively integrates mixing, reaction, separation, catalysis, and detection steps into one chip, significantly simplifying the experimental operation, and using a smartphone for image acquisition and analysis avoids the use of expensive optical analysis instruments, making it applicable to on-site rapid detection.

[0085] The size of the flow channel is also optimized in the present application, and the details are as follows:

[0086] PB buffer is taken as the test object, and the flow channels with diameters of 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm and 3.5 mm are optimized. According to the flow rate of PB under the action of gravity, 3 mm is selected as the flow channel size for subsequent use.

[0087] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are commercially available goods.

[0088] Example 1: A colorimetric biosensor based on a gravity-driven microfluidic chip and Fe-N-C nanozyme signal amplification and its application in rapid detection of foodborne pathogenic bacteria Salmonella typhimurium

[0089] As shown in Figure 1 , the present application provides a gravity-driven microfluidic chip which is combined with a colorimetric biosensor for rapid and sensitive detection of Salmonella typhimurium. The sensor includes two parts: (1) a storage chamber for storage of bacterial samples, washing liquid, waste liquid and other reagents; (2) the design of air holes, which controls the flow of liquid in the chamber through the opening and closing of the air holes of each chamber. The detection process is as shown in Figure 3 , the immunomagnetic beads (polyclonal antibodies for preparing immunomagnetic beads are purchased from Biosynth brand, item number 20C-CR7100RP), bacterial samples and immunomagnetic Fe-N-C nanozyme (monoclonal antibodies for preparing immunomagnetic Fe-N-C nanozyme are purchased from Meridian brand, item number C86309M) are injected into the microfluidic chip at the same time, and the weak magnetic small iron balls are used for mixing and incubation to form a double-antibody sandwich complex of magnetic beads-bacteria-nanoflower, which is released to the separation chamber through the control of liquid by air holes. The double-antibody sandwich complex is captured to the surface by the small magnetic balls pre-embedded in the separation chamber, and after the supernatant is released to the waste liquid chamber by the communication device, the separation and enrichment of the target bacteria are realized; then, the washing liquid in the release chamber 3 is used to wash the non-specific adsorption on the surface of the magnetic beads, and the supernatant is released to the waste liquid chamber; finally, the TMB-H2O2 system in the release chamber 2 is used to catalyze the color change of the complex by the Fe-N-C nanozyme, and after a period of reaction, the catalytic product is transferred to the detection chamber, and the product image is collected and analyzed by using a smartphone image analysis App, realizing the quantitative detection of Salmonella. The biosensor innovatively integrates the steps of mixing, reaction, separation, catalysis and detection into one chip, significantly simplifying the experimental operation, and using a smartphone for image collection and analysis, avoiding the use of expensive optical analysis instruments, so that it can be applied to on-site rapid detection.

[0090] Figure 1 The middle positioning hole is used for aligning the two PDMS pieces when the microfluidic chip is bonded.

[0091] As shown in Figure 4 , good catalytic activity is very important to improve the sensitivity of the sensor. Therefore, the present application uses Fe-N-C with different concentrations (3.9 μg / mL-500 μg / mL) to catalyze 200 μL of TMB-H2O2, and measures the absorbance of the catalytic product at 652 nm. As shown in the figure, as the concentration of nanoscale enzyme increases from 3.9 μg / mL to 500 μg / mL, the absorbance of the catalytic product increases from 0.3 to 2.25, and shows a good linear relationship in the range of 3.9-62.5 μg / mL, indicating that the Fe-N-C nanoscale enzyme has good catalytic ability.

[0092] As shown in Figure 5 , the amount of immunomagnetic beads has a great influence on the capture efficiency of target bacteria, so different amounts of immunomagnetic beads are used to isolate target bacteria from 200 μL of samples with a target bacteria concentration of 2.6 x 10 3 CFU / mL. As shown in Figure 5 , when the amount of immunomagnetic beads increases from 10 μg to 40 μg, the capture efficiency increases significantly from 76.55% to 94.69% within the same capture time. However, further increasing to 50 μg only causes an increase of less than 0.5% in capture efficiency. Therefore, the optimal amount of immunomagnetic beads used in the present application is 30 μg.

[0093] As shown in Figure 6 , the mixing frequency and incubation time are the key to forming the immunomagnetic bead-bacteria-nanoscale enzyme complex. Therefore, 200 μL of Salmonella with a concentration of 10 3 CFU / mL is mixed with 40 μg of immunomagnetic beads with a particle size of 180 nm under different mixing frequencies and incubation times, and the capture efficiency is calculated using the plate count method to evaluate the mixing performance of the small iron balls. As shown in the figure, when the mixing frequency increases from 10 times per minute to 60 times per minute, and the incubation time is 5 to 10 min, the capture efficiency increases from 53.45% to 94.55%. When the mixing frequency is 60 times per minute, and the incubation time is 15 min, the capture efficiency can reach 96.71%. Even if the incubation time is extended, although the capture efficiency is slightly improved, the high capture efficiency of 94.55% is sufficient to ensure the sensitivity of the biosensor. Therefore, the present application selects a mixing frequency of 60 times per minute and an incubation time of 10 min as the optimal mixing conditions.

[0094] As shown in Figure 7As shown, the amount of immune Fe-N-C mimetic enzyme is an important parameter of the biosensor. The present application mixes 200 μL of Salmonella with a concentration of 10 4 CFU / mL with different amounts of immune Fe-N-C mimetic enzyme, mixes at a frequency of 60 times per minute, and incubates for 10 min to form a complex, then uses 100 μL of TMB-H2O2 to catalyze for 15 min. The Fe-N-C mimetic enzyme on the surface of the complex will catalyze the substrate to produce a significant blue change, then uses a mobile phone to take a picture to collect the catalytic product image, and uses software ImageJ to convert it from RGB color space to HSV (Hue-Saturation-Value) color space. The present application uses the saturation of the image to analyze the catalytic results. As shown in Figure 7 When the amount of immune Fe-N-C mimetic enzyme increases from 10 μg to 40 μg, the saturation increases from 124 to 207, and when the amount further increases to 50 μg, the saturation level remains almost unchanged. This shows that 40 μg of immune Fe-N-C mimetic enzyme is sufficient to label the target bacteria. Therefore, the present application selects 40 μg as the optimal immune Fe-N-C mimetic enzyme.

[0095] As shown in Figure 8 In addition, the catalytic time of the mimetic enzyme is also important for the sensitivity of the biosensor. The present application mixes 200 μL of Salmonella with a concentration of 10 4 CFU / mL with 40 μg of immune Fe-N-C mimetic enzyme at a frequency of 50 times per minute, incubates for 5 min to form a magnetic bead-bacteria-Fe-N-C mimetic enzyme complex, and after washing in the separation chamber, adds 100 μL of TMB-H2O2 to react. As shown in Figure 8 With the catalytic time increasing from 5 min to 15 min, the saturation increases from 125 to 196, and when the catalytic time further increases to 20 min, the saturation does not increase significantly. Therefore, the present application selects 15 min as the optimal catalytic time of the mimetic enzyme.

[0096] As shown in Figure 9 This figure is the standard curve of the sensor. Under the optimal conditions, the present application uses bacteria with a concentration of 7.4×10 1 -7.4×10 6 CFU / mL as target bacteria, and performs three repeated tests to explore the relationship between the saturation (S) of the catalytic product and the concentration (C) of the bacteria. With the concentration of the bacteria increasing from 7.4×10 1 CFU / mL to 7.4×10 6 CFU / mL, the saturation increases from 105 to 237. The linear relationship expression is: S=26.3825×lg(C)+60.3875 (R2 According to the three times signal-to-noise ratio, the detection limit of the biosensor is calculated to be 6.8 CFU / 200 μL (the sample volume is 200 μL), that is, the biosensor can detect 7 Salmonella in each sample.

[0097] To verify the specificity of the colorimetric biosensor, the present application detects 2.8×10 4 CFU / mL of target bacteria (Salmonella typhimurium) and 1.7×10 6 CFU / mL of Escherichia coli O157:H7 and 2.5×10 6 CFU / mL of Listeria monocytogenes and two non-target bacteria, and a blank control group. As shown in Table 1, the saturation of the target bacteria and the mixed bacteria is obviously higher than that of the other two non-target bacteria, which indicates that the biosensor has good specificity. Figure 10

[0098] To further explore the influence of finger pressing operation on the detection of the biosensor, four experimental personnel respectively use the biosensor to conduct parallel experiments on Salmonella with a concentration of 2.8×10 4 CFU / mL after receiving simple training. As shown in Table 2, the standard deviation of the detection results of the four persons is less than 6.70%, which indicates that manual operation has no obvious influence on the detection results. Figure 10

[0099] To verify the practicability of the biosensor, the present application adds Salmonella with a concentration of 2.8×10 2 -2.8×10 5 CFU / mL to the supernatant of pork, and simultaneously detects and verifies by using the biosensor and the gold standard culture method. As shown in Table 1, the recovery rates of the biosensor for target bacteria with a concentration of 2.8×10 2 -2.8×10 5 CFU / mL are 87.14%, 90.78%, 128.86% and 120.94% respectively, and the relative standard deviations are all less than 7.5%. The results show that the method has good practicability for the detection of Salmonella in actual pork samples. However, since the real pork sample contains complex backgrounds such as fat, protein, blood, etc., it has some influence on the immune reaction between the target bacteria and the simulation enzyme, and the recovery rate still has certain fluctuation. In addition, these impurities can non-specifically adsorb part of the immune Fe-N-C simulation enzyme, which reduces the number of immune Fe-N-C simulation enzyme reacting with the target bacteria, and thus reduces the detection signal.

[0100] Table 1 Determination of Salmonella typhimurium recovery in spiked samples and recovery results ​​

[0101]

[0102] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. It is therefore intended that whatever lies within the scope of the application be covered by the appended claims and that there be taken as a patent or patents thereon.

Claims

1. A method for constructing a colorimetric biosensor based on Fe-NC nanozyme signal amplification, characterized in that, Includes the following steps: (1) Preparation of Fe-NC nanozymes; (2) Preparation of amination-modified Fe-NC nanozymes; (3) Preparation of immunonanozymes, namely aminated Fe-NC nanozymes coupled with primary antibodies; (4) Preparation of immunomagnetic beads, i.e., magnetic beads coupled with a second antibody; Further, the preparation method of the Fe-NC nanozyme in step (1) includes: a. Mix Zn(NO3)2·6H2O, Fe(NO3)3·9H2O with methanol to obtain solution A; b. Mix 2-methylimidazole with methanol to obtain solution B; c. Mix solution A and solution B to obtain Fe-ZIF-8 precursor; finally, pyrolyze at 800-900°C with N2 to obtain Fe-NC nanozyme; The molar ratio of Zn(NO3)2·6H2O to Fe(NO3)3·9H2O is (11-15):1, and the molar ratio of 2-methylimidazolium to zinc ions is (10-12):

1. Step (3) The first antibody and step (4) The second antibody are different antibodies against the same pathogen, and the first antibody and the second antibody bind to different epitopes of the pathogen, respectively.

2. The method according to claim 1, characterized in that, The preparation method of the aminated Fe-NC nanozyme in step (2) includes: Fe-NC nanozymes were dissolved in anhydrous ethanol, APTES were added, and the pH was adjusted to 10.5-11.0 with ammonia. The mixture was then stirred in a water bath at 50℃-65℃ for 5-8 hours to obtain aminated Fe-NC nanozymes.

3. The method according to claim 1, characterized in that, The pathogenic bacterium is Salmonella; The first antibody is a Salmonella monoclonal antibody, and the second antibody is a Salmonella polyclonal antibody.

4. The method according to claim 3, characterized in that, Step (3) includes: mixing Salmonella monoclonal antibody with aminated Fe-NC nanozyme, adding 5%-10% BSA to block for 30 min, and finally reconstitute with 1%-3% BSA to obtain immunonanozyme.

5. The method according to claim 3, characterized in that, Step (4) includes: adding a mixture of EDC and NHSS to the magnetic beads, activating for 1 h to obtain immunomagnetic beads; after magnetic separation and recovery of the magnetic beads, adding Salmonella polyclonal antibodies and conjugating for 2-4 h; adding blocking solution to the conjugated product for blocking, magnetic separation and recovery of the magnetic beads, and reconstitution solution for rehydration.

6. A microfluidic chip for a colorimetric biosensor based on Fe-NC nanozyme signal amplification, characterized in that, It is a gravity-driven microfluidic chip, including an inlet port, an air vent, several flow channels, and the following six chambers: a reaction chamber, a cleaning solution chamber, a TMB-H2O2 system chamber, a separation chamber, a waste liquid chamber, and a detection chamber; wherein, the reaction chamber is pre-embedded with weakly magnetic iron balls, and the capture rate is improved by mixing the sample, immunomagnetic beads, and immunonanozymes with the weakly magnetic iron balls; wherein, the immunomagnetic beads and immunonanozymes are the same as those in the method of any one of claims 1-5.

7. The application of the microfluidic chip of claim 6 in the qualitative and quantitative detection of pathogenic bacteria in samples.

8. A method for detecting pathogenic bacteria based on a colorimetric biosensor and microfluidic chip using Fe-NC nanozyme signal amplification, characterized in that, Detecting pathogenic bacteria in samples using the microfluidic chip described in claim 6; Includes the following steps: Immunomagnetic beads, sample solution, and immunonanozyme are pre-mixed and injected into the reaction chamber of a microfluidic chip. After a period of mixing and reaction using an external magnet to agitate the weakly magnetic iron balls, the mixture is released through a flow channel into the separation chamber via a vent switch. The resulting dual-antibody sandwich complex is captured by the built-in NdFeB microspheres. The supernatant is released into the waste liquid chamber via vent opening control. Then, the washing liquid in the washing liquid chamber is released to clean the non-specific adsorption on the surface of the magnetic beads, and the supernatant is released into the waste liquid chamber. Next, the TMB-H2O2 system in the TMB-H2O2 system chamber is released into the separation chamber. The chromogenic substrate is catalyzed by the immunonanozyme on the dual-antibody sandwich complex. After the catalytic reaction is complete, the mixture is released into the detection chamber. In the detection chamber, a smartphone image analysis app is used to analyze the catalytic products to determine the concentration of pathogenic bacteria in the sample.

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