Microorganism separation detection system and detection method

文档序号：188740 发布日期：2021-11-02 浏览：37次中文

阅读说明：本技术 微生物分离检测系统及检测方法 (Microorganism separation detection system and detection method ) 是由林建涵霍晓婷王蕾戚武振汪思源段宏于 2021-07-16 设计创作，主要内容包括：本发明提供一种微生物分离检测系统及检测方法。所述系统包括圆环形微流控芯片、旋转磁场装置、连接元件、步进电机、步进电机驱动器、固定装置、嵌入式装置和智能手机图像分析设备等。本发明提供的微生物分离检测系统集分离、标记、洗涤、催化、检测于一体,具有操作自动化、反应速度快、体积小等特点,在食源性致病菌、动物病毒等病原微生物快速检测方面具有广阔的应用前景。(The invention provides a microorganism separation detection system and a detection method. The system comprises a circular micro-fluidic chip, a rotating magnetic field device, a connecting element, a stepping motor driver, a fixing device, an embedded device, an intelligent mobile phone image analysis device and the like. The microorganism separation and detection system provided by the invention integrates separation, marking, washing, catalyzing and detecting, has the characteristics of automation in operation, high reaction speed, small volume and the like, and has wide application prospect in the aspect of rapid detection of pathogenic microorganisms such as food-borne pathogenic bacteria, animal viruses and the like.)

1. The microorganism separation detection system is characterized by comprising a circular microfluidic chip, a rotating magnetic field device, a connecting element, a stepping motor driver, a fixing device, an embedded device and intelligent mobile phone image analysis equipment;

the annular micro-fluidic chip is provided with n spindle-shaped liquid storage cavities and arc-shaped reaction cavities connected with outlets of the liquid storage cavities, the liquid storage cavities are used for storing loaded liquid, and the arc-shaped reaction cavities are used for separating and detecting target microorganisms; wherein n is an integer greater than or equal to 2;

the rotating magnetic field device is connected with the circular microfluidic chip in an embedded mode through the connecting element, and magnetic materials loaded in the circular microfluidic chip can move step by step under the driving of a magnetic field; the connecting element comprises a central shaft, a bearing and a shaft support;

two L-shaped rotating shafts contained in the rotating magnetic field device can perform circular motion under the driving of the stepping motor;

the stepping motor driver controls the stepping motor to operate;

the fixing device is a three-layer bracket, the upper layer is detachably fixed with a circular micro-fluidic chip and a rotating magnetic field device which are in embedded connection, the middle layer is detachably fixed with a stepping motor, and the lower layer is detachably fixed with a stepping motor driver and an embedded device; wherein, the embedded device is a singlechip; the embedded connection annular micro-fluidic chip and the rotating magnetic field device are taken as a whole and are detachably connected with the stepping motor through the shaft support;

the structure of the circular micro-fluidic chip is as follows:

the circular microfluidic chip is a copper coin structure with a circular hole in the center, and the diameter of the circular hole is larger than or equal to that of a cylindrical magnet in the rotating magnetic field device, so that the circular microfluidic chip can smoothly penetrate through the cylindrical magnet;

the annular microfluidic chip is formed by bonding an annular PDMS (polydimethylsiloxane) containing an annular microchannel and an annular glass sheet which are formed by reverse molding;

the arc reaction cavity is separated by 2n air outlets arranged on the annular microchannel to form n independent open arc cavities, and two air outlets are respectively arranged at two ends of the inner side of each cavity;

n spindle-shaped liquid storage cavities which are radially arranged at the center are arranged on the circular micro-fluidic chip, and the included angle between the liquid storage cavities is 360 degrees/n;

one end of each liquid storage cavity, which is close to the center of the chip, is provided with a sample inlet, the other end of each liquid storage cavity is provided with an outlet, and the outlet of each liquid storage cavity is communicated with the arc-shaped reaction cavity;

the rotating magnetic field device comprises a cylindrical magnet, two L-shaped rotating shafts, a central shaft, two bearings and a fixing ring;

the two L-shaped rotating shafts are respectively fixed at two ends of the cylindrical magnet through a central shaft penetrating through the cylindrical magnet, the center of the cylindrical magnet is hollow, and the central shaft nested bearing can just penetrate through the hollow;

the central shaft is divided into an upper half shaft, a middle shaft and a lower half shaft; the length of the lower half shaft is greater than that of the upper half shaft; the upper half shaft fixes one L-shaped rotating shaft to one end of the cylindrical magnet through the opening formed in the tail part of the long end of the rotating shaft, and the lower half shaft fixes the other L-shaped rotating shaft to the other end of the cylindrical magnet through the opening formed in the tail part of the long end of the rotating shaft;

the two L-shaped rotating shafts have the same size; the tail part of the short end of the L-shaped rotating shaft is of a semicircular contraction structure and is used for gathering magnetic induction lines;

the annular micro-fluidic chip is nested in the middle of the cylindrical magnet, one L-shaped rotating shaft is positioned above the chip, the other L-shaped rotating shaft is positioned below the chip, an air gap is formed between the tail parts of the short ends of the two L-shaped rotating shafts, and the height of the air gap is greater than the thickness of the chip; a vertical magnetic field is generated at the air gap and correspondingly acts on an arc-shaped reaction cavity on the chip;

the lower half shaft penetrating through the central shaft of the cylindrical magnet is connected with a stepping motor shaft through a shaft support;

the fixing ring is used for fixing the cylindrical magnet, so that the cylindrical magnet does not rotate when the L-shaped rotating shaft rotates, and the stepping motor can drive the L-shaped rotating shaft to rotate through the central shaft;

the embedded device is used for controlling the reaction time and the reaction speed;

the intelligent mobile phone image analysis equipment is used for detecting a reaction result.

2. The system of claim 1, wherein the L-shaped rotating shafts are made of iron, and two small bar-shaped magnets are arranged at the tail part of the short end of each L-shaped rotating shaft to enhance the magnetic field.

3. The system of claim 2, wherein the central shaft, the shaft holder, and the retaining ring are each a non-magnetic material or a magnetically permeable material.

4. The system of claim 1, wherein the embedded device is a single-chip microcomputer;

the stepping motor driver receives the pulse signal from the singlechip and controls the stepping motor to move according to the set direction.

5. The system according to any one of claims 2 to 4, wherein the inner diameter of the circular microfluidic chip is 18mm and the outer diameter is 64 mm; 6 spindle-shaped liquid storage cavities with the same size and shape are arranged on the chip; the diameter of the liquid storage cavity sample inlet is 0.8 mm; the diameter of an air outlet arranged on the annular microchannel is 0.6 mm;

the dimensions of the hollow cylindrical magnet are: the outer diameter is 18mm, the inner diameter is 8mm, and the height is 28 mm;

the dimensions of the L-shaped rotating shaft are as follows: the length is 38.5mm, the width is 5mm, and the height is 17 mm; the tail part of the long end of the L-shaped rotating shaft is provided with a hole with the aperture of 3mm, and the upper half shaft and the lower half shaft of the central shaft can penetrate through the hole to fix the L-shaped rotating shaft on the cylindrical magnet;

the size of the small bar-shaped magnet is as follows: 12.7mm × 6.4mm × 3.2 mm;

the central shaft is made of photosensitive resin; the total length of the central shaft is 46 mm; the radius of the middle shaft is 2.5mm, and the length is 28 mm; the radius of the upper half shaft is 1.5mm, and the length is 3 mm; the radius of the lower half shaft is 1.5mm, and the length is 15 mm;

the bearing is made of iron; the size is as follows: the outer diameter is 8mm, the inner diameter is 5mm, and the height is 2 mm;

the shaft support is made of photosensitive resin and comprises an upper support shaft, a lower support shaft and a central solid part connected with the upper support shaft and the lower support shaft, wherein the upper support shaft is used for nesting a lower half shaft of the central shaft, and the lower support shaft is used for nesting a shaft of the stepping motor; the radius of the upper supporting shaft is 3mm, the length of the upper supporting shaft is 7mm, and the radius of the opening is 1.5 mm; the radius of the lower support shaft is 4mm, the length of the lower support shaft is 10mm, and the radius of the opening is 2.5 mm.

6. The method for separating and detecting microorganisms using the system of claim 5, wherein the method comprises:

s1, injecting the modified immune nano nickel wire solution, the to-be-detected sample solution containing the target microorganism, the HRP-nanoflower solution, the cleaning solution 1, the cleaning solution 2 and the TMB color development solution into each spindle-shaped liquid storage cavity from the sample injection hole in the clockwise or counterclockwise direction of the circular microfluidic chip respectively; then the liquid in the liquid storage cavity enters the peripheral arc reaction cavity through centrifugal force to form independent liquid sections;

s2, placing the chip forming the liquid segment in a rotating magnetic field, and carrying out the following operations:

starting a stepping motor to drive an L-shaped rotating shaft to circularly move in a nano nickel wire liquid section to form a nano nickel wire net;

driving the nano nickel wire net to enter a sample liquid section containing target microorganisms to be detected by the L-shaped rotating shaft to circularly move, and capturing the target microorganisms on the nano nickel wire net to form magnetic microorganisms;

driving the magnetic microorganisms to enter an HRP-nanoflower liquid section for circular movement by the L-shaped rotating shaft, and carrying out microorganism marking to form nanoflower microorganisms;

driving the nanoflower microorganisms to enter a liquid section of the cleaning solution 1 for circular movement by the L-shaped rotating shaft and then enter a liquid section of the cleaning solution 2 for circular movement;

the cleaned nanoflower microorganisms enter a TMB color development liquid section to circularly move under the drive of an L-shaped rotating shaft to perform enzyme catalysis reaction;

driving the nanometer flower microbe to enter the initial nanometer nickel wire liquid segment with L-shaped rotating shaft to stop reaction and stop the motor;

and after the reaction is finished, analyzing the color of the product by using intelligent mobile phone image analysis equipment, thereby realizing qualitative and quantitative detection of the target microorganism.

7. The method of claim 6, wherein the modified immunonickel nanowire solution is prepared by a method comprising:

(1) 75 mu L of 1M nickel chloride hexahydrate solution and 15mL of ethylene glycol are mixed and added into a beaker and heated to 100 ℃;

(2) dropwise adding 0.5mL of hydrazine monohydrate to the mixture, and keeping the temperature at 100 ℃;

(3) heating was continued for 30min until a dark grey product formed and floated on the surface of the solution;

(4) collecting the generated dark gray product, namely the nano nickel wire, and washing for 3 times by using deionized water in combination with magnetic separation, and washing for 2 times by using absolute ethyl alcohol in combination with magnetic separation;

(5) dispersing the cleaned nano nickel wire in ethanol containing 0.03% w/v polyvinylpyrrolidone to obtain 1mg/mL nano nickel wire solution;

(6) ultrasonically and uniformly mixing the nano nickel wire solution to form a uniform dispersion system; adding 500 mu L of nano nickel wire solution into a reaction bottle, and cleaning for 2 times by using ultrapure water;

(7) will 100μL H₂O₂、100μL NH₄OH and 500 mu L of deionized water are added into the cleaned nano nickel wire, and the mixed solution is placed in a water bath at 80 ℃ for 30min to obtain a nano nickel wire modified with hydroxyl;

(8) recovering the hydroxyl-modified nano nickel wire by using a magnetic frame, cleaning the nano nickel wire by using ultrapure water for 1 time and absolute ethyl alcohol for 1 time, and then placing the nano nickel wire in an oven for drying;

(9) preparing a 1% [3- (methoxylsilyl) propyl ] succinic anhydride solution by using absolute ethyl alcohol, resuspending the dried hydroxyl-modified nano nickel wire by using the 1% [3- (methoxylsilyl) propyl ] succinic anhydride solution, and then placing the nano nickel wire on a blending instrument for overnight reaction at room temperature to obtain a carboxyl-modified nano nickel wire;

(10) recovering the carboxyl modified nano nickel wire by using a magnetic frame the next day, washing the nano nickel wire by using absolute ethyl alcohol for 3 times, then washing the nano nickel wire by using ultrapure water for 2 times, then adding 500 mu L of NaOH solution with 1M and pH 9, and reacting for more than 5 hours;

(11) washing the carboxyl-modified nano nickel wire which is resuspended by the alkali liquor for 3 times by using ultrapure water, and then washing for 1 time by using a PB solution with the pH of 7.4;

(12) adding 500 mu g of EDC and NHSS, reacting for 1h, and activating carboxyl;

(13) washing the nano nickel wire modified with the activated carboxyl for 2 times by using a PB solution with pH of 7.4;

(14) redissolving and modifying the nano nickel wire with the activated carboxyl by using 420 mu L of PB solution with pH 7.4, then adding 41.67 mu L of 1.2mg/mL streptavidin solution and 200 mu g skim milk, and reacting for 1h at room temperature;

(15) washing the nano nickel wire modified with the activated carboxyl by using a PB solution with the pH of 7.4 for 2 times, adding 500 mu L of 1% skim milk, and sealing for 1h to obtain a streptavidin nickel wire;

(16) adding 60 mu g of streptavidin nickel wire into 500 mu L of PBS solution, carrying out magnetic separation and cleaning for 2 times, and removing the skim milk solution;

(18) and (5) carrying out magnetic separation and cleaning to remove redundant unbound antibodies, thus obtaining the antibody.

8. The method of claim 7, wherein the preparation method of the HRP-nanoflower solution comprises:

(1) 20 mu g of detection antibody solution with the concentration of 3.35mg/mL and 180 mu g of 5mg/mL HRP enzyme solution for the target microorganism are added into 1mL of 3mM PBS solution;

(2) then 20. mu.L of 200mM CaCl₂Adding the solution into the mixed solution obtained in the step (1), incubating at room temperature for 12h, centrifuging at 15000rpm for 5min, collecting precipitate, washing with ultrapure water for 3 times, centrifuging to obtain HRP-nano flower material, and re-dissolving with 400 μ L of ultrapure water to obtain the final product.

9. The method according to claim 7, wherein the cleaning solutions 1 and 2 are PBST solutions.

10. The method of any one of claims 7-9, wherein the target microorganism is salmonella typhimurium;

in the step S1, the sample adding amount of each liquid storage cavity is 20 mu L; the centrifugation conditions were: centrifuging at 650-700 rpm for 3 s.

Technical Field

The invention relates to the technical field of microorganism detection, in particular to a microorganism separation detection system and a detection method.

Background

Food-borne pathogenic bacteria have been considered as the most major risk factors for food safety. Therefore, the development of the site screening of the food-borne pathogenic bacteria has important significance for preventing the outbreak of the food-borne diseases.

At present, the common food-borne pathogenic bacteria separation method is mainly immune nano magnetic separation. However, this method requires manual operation. The immune magnetic grid/mesh separation is a separation mode which utilizes a special magnetic field design to enable magnetic materials to be distributed in a chain shape in a channel, so that the collision and combination probability of bacteria and the magnetic materials is increased. The separation mode can effectively improve the capture efficiency and realize automatic operation by combining with the microfluidic chip. In the national standard, the methods for detecting food-borne pathogenic bacteria include three methods, namely culture method, polymerase chain reaction and enzyme-linked immunosorbent assay. However, none of the three methods can achieve field detection. The biosensor is a food-borne pathogenic bacteria detection technology which develops rapidly in recent decades, and has the characteristics of simple and convenient operation, low cost, high sensitivity, strong specificity, high response speed, capability of on-site detection and the like. Therefore, the development of a platform and a method for detecting food-borne pathogenic bacteria by utilizing immunomagnetic net separation and a biosensor is of great significance for field detection.

Disclosure of Invention

The invention aims to provide a microorganism separation detection system and a detection method.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a microorganism separation detection system, which includes a circular microfluidic chip, a rotating (high gradient) magnetic field device, a connection element, a stepping motor driver, a fixing device, an embedded device, and a smartphone image analysis apparatus.

The annular micro-fluidic chip is provided with n spindle-shaped liquid storage cavities and arc-shaped reaction cavities (the arc-shaped reaction cavities are arranged at the outer edge of the chip) connected with outlets of the liquid storage cavities, the liquid storage cavities are used for storing loaded liquid, and the arc-shaped reaction cavities are used for separating and detecting target microorganisms; wherein n is an integer of 2 or more (preferably n is 6);

two L-shaped rotating shafts contained in the rotating magnetic field device can perform circular motion under the driving of the stepping motor;

the stepping motor driver controls the stepping motor to run (the stepping motor driver receives a pulse signal from the singlechip and controls the stepping motor to move according to a set direction);

the fixing device is a three-layer bracket, a circular micro-fluidic chip and a rotating magnetic field device which are connected in an embedded mode are detachably fixed on the upper layer, a stepping motor is detachably fixed on the middle layer, and a stepping motor driver and an embedded device (a single chip microcomputer is the embedded device) are detachably fixed on the lower layer; the embedded connection annular micro-fluidic chip and the rotating magnetic field device are taken as a whole and are detachably connected with the stepping motor through the shaft support;

the structure of the circular micro-fluidic chip is as follows:

the annular microfluidic chip is formed by bonding an annular PDMS (polydimethylsiloxane) containing an annular microchannel and an annular glass sheet which are formed by reverse molding; the chip is sleeved in the middle of the cylindrical magnet by setting a proper size, and the chip comprises 1.5mm thick PDMS and 0.5mm thick glass sheet, so that the chip can be kept parallel/balanced after being placed in the central position of the cylindrical magnet by virtue of the thickness of the chip;

the arc reaction cavity is separated by 2n air outlets arranged on the annular microchannel to form n independent open arc cavities (used for forming independent liquid sections), and two air outlets are respectively arranged at two ends of the inner side of each cavity (namely the number of the liquid storage cavities is equal to the number of the arc cavities is equal to the number of the 1/2 air outlets);

n spindle-shaped liquid storage cavities which are radially (uniformly) arranged at the center are arranged on the circular micro-fluidic chip, and the included angle between the liquid storage cavities is 360 degrees/n;

the rotating magnetic field device comprises a cylindrical magnet, two L-shaped rotating shafts, a central shaft, two bearings and a fixing ring;

the annular micro-fluidic chip is nested in the middle of the cylindrical magnet, one L-shaped rotating shaft is positioned above the chip, the other L-shaped rotating shaft is positioned below the chip, an air gap is formed between the tail parts of the short ends of the two L-shaped rotating shafts, and the height of the air gap is greater than the thickness of the chip; a vertical (high gradient) magnetic field is generated at the air gap and correspondingly acts on an arc-shaped reaction cavity on the chip;

the lower half shaft penetrating through the central shaft of the cylindrical magnet is connected with a stepping motor shaft through a shaft support;

the fixing ring is used for fixing the cylindrical magnet, when the L-shaped rotating shaft rotates, the cylindrical magnet does not rotate, and the stepping motor can drive the L-shaped rotating shaft to rotate through the central shaft and the shaft support.

The embedded device is used for controlling the reaction time and the reaction speed.

The intelligent mobile phone image analysis equipment is used for detecting a reaction result. Preferably, the image analysis device is developed by using an android phone platform.

Preferably, the L-shaped rotating shafts are made of iron, and two small bar-shaped magnets are further arranged at the tail part of the short end of each L-shaped rotating shaft to enhance the magnetic field.

Preferably, the central shaft, the shaft support and the fixing ring are all made of non-magnetic materials or magnetic materials.

In one embodiment of the present invention, in the microorganism separation and detection system: the inner diameter of the annular micro-fluidic chip is 18mm, and the outer diameter of the annular micro-fluidic chip is 64 mm; 6 spindle-shaped liquid storage cavities with the same size and shape are arranged on the chip; the diameter of the liquid storage cavity sample inlet is 0.8 mm; the diameter of an air outlet arranged on the annular microchannel is 0.6 mm;

the dimensions of the hollow cylindrical magnet (preferably N52) are: the outer diameter is 18mm, the inner diameter is 8mm, and the height is 28 mm;

the dimensions of the small bar magnet (preferably N52) are: 12.7mm × 6.4mm × 3.2 mm;

the bearing is made of iron; the size is as follows: the outer diameter is 8mm, the inner diameter is 5mm, and the height is 2 mm;

In a second aspect, the present invention provides a method for separately detecting microorganisms using the microorganism separation detection system, the method comprising:

s1, injecting the modified immune nano nickel wire solution, the to-be-detected sample solution containing the target microorganism, the HRP-nano flower solution (immune nano flower solution), the cleaning solution 1, the cleaning solution 2 and the TMB color development solution (commercial TMB color development solution) into each spindle-shaped liquid storage cavity from the sample injection hole in the clockwise or anticlockwise direction of the circular microfluidic chip respectively; then the liquid in the liquid storage cavity enters the peripheral arc reaction cavity through centrifugal force to form independent liquid sections;

s2, placing the chip forming the liquid segment in a rotating (high gradient) magnetic field, and performing the following operations:

starting a stepping motor to drive an L-shaped rotating shaft to circularly move in a nano nickel wire liquid section to form a nano nickel wire net;

the cleaned nanoflower microorganisms enter a TMB color development liquid section to circularly move under the drive of an L-shaped rotating shaft to perform enzyme catalysis reaction;

driving the nanometer flower microbe to enter the initial nanometer nickel wire liquid segment with L-shaped rotating shaft to stop reaction and stop the motor;

Preferably, the preparation method of the modified immune nano nickel wire solution comprises the following steps:

(1) 1M Nickel chloride hexahydrate solution (99.9% pure) 75 μ L and ethylene glycol (99.8%) 15mL were mixed and added to a beaker and heated to 100 ℃;

(2) dropwise adding 0.5mL of hydrazine monohydrate to the mixture, and keeping the temperature at 100 ℃;

(3) heating was continued for 30min until a dark grey product formed and floated on the surface of the solution;

(5) dispersing the cleaned nano nickel wire in ethanol containing 0.03% w/v polyvinylpyrrolidone to obtain 1mg/mL nano nickel wire solution;

(6) ultrasonically and uniformly mixing the nano nickel wire solution to form a uniform dispersion system; adding 500 μ L of nano nickel wire solution into a reaction bottle, and cleaning with ultrapure water for 2 times (to remove the protective agent-ethanol on the surface of the nickel wire);

(7) 100 mu L H₂O₂、100μL NH₄OH and 500 mu L of deionized water are added into the cleaned nano nickel wire (hydroxylation modification of the nano nickel wire is carried out), and the mixed solution is placed in a water bath at 80 ℃ for 30min to obtain the nano nickel wire modified with hydroxyl (the reaction bottle is not covered);

(8) recovering the hydroxyl-modified nano nickel wire by using a magnetic frame, cleaning the nano nickel wire by using ultrapure water for 1 time, cleaning the nano nickel wire by using absolute ethyl alcohol for 1 time, and then drying the nano nickel wire in an oven (drying the nano nickel wire for about 8min at 65 ℃);

(9) preparing a 1% [3- (methoxylsilyl) propyl ] succinic anhydride solution by using absolute ethyl alcohol, resuspending the dried hydroxyl-modified nano nickel wire (carrying out carboxylation modification on the nano nickel wire) by using a 1% [3- (methoxylsilyl) propyl ] succinic anhydride (molecular formula C10H18O6Si) solution (10 mu L of [3- (methoxylsilyl) propyl ] succinic anhydride +990 mu L of absolute ethyl alcohol), and then placing the nano nickel wire on a mixing machine for overnight reaction at room temperature for 15H to obtain the carboxyl-modified nano nickel wire;

the structure of the [3- (methoxysilyl) propyl ] succinic anhydride is shown as the formula I):

(10) the next day, the nano nickel wire modified with the carboxyl is recovered by a magnetic frame, washed 3 times by absolute ethyl alcohol (to remove residual carboxylation reagent on the surface), washed 2 times by ultrapure water (to remove residual ethyl alcohol), and then added with 1M NaOH solution (pH 9, pH adjusted by HCl) 500 microliter to react for more than 5 hours (the water solubility of the nickel wire solution can be observed by naked eyes to be good, namely the carboxyl is fully opened);

(11) washing the carboxyl-modified nano nickel wire which is resuspended by the alkali liquor for 3 times by using ultrapure water (so as to remove residual NaOH), and then washing for 1 time by using a PB solution with the pH value of 7.4;

(12) add 500. mu.g EDC (10mg/mL, dissolved in PB solution at pH 7.4) and NHSS (10mg/mL, dissolved in PB solution at pH 7.4) and react for 1h to activate the carboxyl groups (500. mu.L final PB solution at pH 7.4);

(13) washing the nano nickel wire modified with the activated carboxyl group 2 times with a PB solution with pH 7.4 (to remove the residual EDC and NHSS);

(14) redissolving and modifying the nano nickel wire with the activated carboxyl by using 420 mu L of PB solution with pH 7.4, then adding 41.67 mu L of 1.2mg/mL streptavidin solution and 200 mu g of skim milk (used for binding the streptavidin and the nickel wire and stabilizing a reaction system), and reacting for 1h at room temperature;

(15) washing the nano nickel wire modified with the activated carboxyl by a PB solution with the pH of 7.4 for 2 times (to remove unbound streptavidin), adding 1% skim milk (dissolved by the PB solution with the pH of 7.4) 500 mu L, and sealing for 1h to obtain a streptavidin nickel wire;

(16) adding 60 mu g of streptavidin nickel wire into 500 mu L of PBS solution, carrying out magnetic separation and cleaning for 2 times, and removing the skim milk solution;

(17) then re-dissolving the nano nickel wire by using 500 mu L of PBS solution, adding 4 mu g of capture antibody, and incubating for 45min at room temperature; wherein the capture antibody specifically recognizes and binds to the target microorganism (when the target microorganism is Salmonella typhimurium, the capture antibody may be a biotinylated polyclonal antibody against Salmonella typhimurium at a concentration of 2.25 mg/mL);

(18) and (5) carrying out magnetic separation and cleaning to remove redundant unbound antibodies, thus obtaining the antibody.

Preferably, the preparation method of the HRP-nanoflower solution comprises the following steps:

(1) 20. mu.g of a detection antibody against the target microorganism (when the target microorganism is Salmonella typhimurium, the detection antibody may be an anti-Salmonella typhimurium monoclonal antibody) at a concentration of 3.35mg/mL, 180. mu.g of a 5mg/mL HRP enzyme solution was added to 1mL of a 3mM PBS solution;

(2) then 20. mu.L of 200mM CaCl₂Adding the solution (prepared by ultrapure water) into the mixed solution in the step (1), incubating at room temperature for 12h (self-assembly to form nanoflower), centrifuging at 15000rpm for 5min, collecting precipitate, washing with ultrapure water for 3 times, centrifuging to obtain HRP-nanoflower material, and re-dissolving with 400 μ L of ultrapure water to obtain the final product.

Preferably, the cleaning solution 1 and the cleaning solution 2 are PBST solutions.

In the present invention, the target microorganism includes, but is not limited to, salmonella typhimurium.

Preferably, the sample adding amount of each liquid storage cavity in the step S1 is 20 μ L; the centrifugation conditions were: centrifuging at 650-700 rpm for 3 s;

when the microorganism of interest is Salmonella typhimurium, the capture antibody can be a biotinylated polyclonal antibody (2.25mg/mL) against Salmonella typhimurium, available from Fitzgerald, USA. The detection antibody may be a monoclonal antibody (3.35mg/mL) against Salmonella typhimurium, available from Abcam, USA.

Compared with the prior art, the microorganism rapid separation detection system/platform provided by the invention has the following advantages:

and (I) the injection of the reaction reagent and the sample to be detected is automated. As long as the sample is stored in the liquid storage cavity of the microfluidic chip in advance, the automatic injection and automatic distribution of all solutions and no cross contamination can be realized by utilizing the centrifugal force.

And (II) putting the sample into a rotating high-gradient magnetic field, starting the stepping motor to rotate for one circle to complete the whole reaction, and is simple and convenient. And the nano nickel wire can form a nano nickel wire net to fill the whole channel section in the presence of a magnetic field, so that the collision and combination probability of an antibody (a capture antibody) on the nano nickel wire and microorganisms can be increased, and the capture efficiency is greatly improved.

And thirdly, carrying out gray level analysis on the reaction result by utilizing the image analysis application developed by the android mobile phone platform, and further uploading the detection result to the cloud platform for risk assessment, thereby realizing risk early warning on microorganisms.

The microorganism separation and detection system integrates separation, marking, washing, catalysis and detection, has the characteristics of automation in operation, high reaction speed, small volume and the like, and has wide application prospect in the aspect of quickly detecting pathogenic microorganisms such as food-borne pathogenic bacteria, animal viruses and the like.

Drawings

FIG. 1 is a schematic structural diagram of a circular microfluidic chip of a microbiological detection platform according to a preferred embodiment of the present invention.

FIG. 2 is a schematic view of the overall structure of the rotating high gradient magnetic field of the microorganism detection platform according to the preferred embodiment of the present invention.

FIG. 3 is a schematic diagram of the structure of the components of the rotating high gradient magnetic field in the preferred embodiment of the present invention.

FIG. 4 is a schematic view of the overall structure of the microorganism detection platform according to the preferred embodiment of the present invention.

FIG. 5 is a schematic diagram illustrating the process of the microorganism detection principle in the preferred embodiment of the present invention.

Fig. 6 is a graph showing the gray level and the HRP-nanoflower concentration obtained by analyzing the image of the smartphone according to the preferred embodiment of the present invention.

Fig. 7 is a calibration curve between the gray-level values obtained by analyzing the images of the smart phone and the target microorganisms with different concentrations in the preferred embodiment of the present invention.

FIG. 8 is a photograph of the constructed microorganism separation and detection system.

FIG. 9 is a diagram illustrating the optimized amount of nano-nickel wires in the preferred embodiment of the present invention.

FIG. 10 is a diagram illustrating the optimization result of the rotating speed of the rotating magnetic field according to the preferred embodiment of the present invention.

FIG. 11 is a graph showing the results of the optimization of the number of capturing cycles of microorganisms in the preferred embodiment of the present invention.

FIG. 12 is a graph showing the optimized results of the amount of the immune nanoflower in the preferred embodiment of the present invention.

FIG. 13 is a graph showing the optimization results of the time period for which the immune nanoflower catalyzes the color development of TMB in the preferred embodiment of the present invention.

FIG. 14 is a graph showing the results of the specificity between the gray-scale values obtained from the analysis of the images of the cell phone and different non-target microorganisms in the preferred embodiment of the present invention.

FIG. 15 is a graph showing the results of the detection between the gray level values obtained from the analysis of the mobile phone image and the chicken samples with different concentrations of the labeled target microorganism in the preferred embodiment of the present invention.

In the figure, 1-sample inlet, 2-liquid storage cavity, 3-arc reaction cavity, 4-gas outlet, 5-circular microfluidic chip, 6-cylindrical magnet, 7-L type rotating shaft, 8-bar small magnet, 9-central shaft, 10-shaft support, 11-bearing, 12-upper half shaft, 13-lower half shaft, 14-fixing ring, 15-rotating magnetic field device, 16-stepping motor, 17-stepping motor driver, 18-single chip (embedded device), 19-fixing device and 20-smart phone image analysis equipment.

Detailed Description

The invention aims to provide a platform/system for rapid separation and detection of microorganisms, so as to solve the problems of cross contamination caused by reagent addition and low capture efficiency in solid-liquid reaction in the prior art.

The invention also provides a rapid microorganism separation and detection method based on the catalytic color development of the nano nickel wire mesh and the HRP-nanoflower.

The invention adopts the following technical scheme:

the platform for the rapid separation and detection of microorganisms comprises: the device comprises a circular micro-fluidic chip, a rotary high-gradient magnetic field, an embedded device, intelligent mobile phone image analysis equipment and a three-layer support.

According to a first aspect of the present invention, there is provided an automatic sample adding method, comprising: the method is characterized in that a circular micro-fluidic chip is manufactured, automatic sample adding and automatic distribution of reaction reagents are realized by utilizing centrifugal force, and cross contamination is avoided; the rotary high gradient magnetic field is utilized to form a nano nickel wire net so as to automatically realize the capture of target microorganisms with high separation efficiency.

The annular micro-fluidic chip is used for storing a reaction reagent and a sample to be detected.

The rotating high gradient magnetic field is used for capturing target microorganisms, labeling signal probes and carrying out enzyme catalytic reaction.

The embedded device is used for controlling the biological reaction time and the reaction speed.

The smartphone image analysis device is used for detecting a product of an enzyme-catalyzed reaction.

The annular microfluidic chip is formed by bonding an annular PDMS (polydimethylsiloxane) containing a microchannel and an annular glass sheet, wherein the annular PDMS is formed by reverse molding. The outer diameter of the chip is the diameter of the whole chip, and the inner diameter is the diameter of the annular opening. The chip comprises n spindle-shaped liquid storage cavities close to the circle center and an arc-shaped reaction cavity connected with the outlets of the liquid storage cavities. The inside border department of arc reaction chamber sets up 2n gas outlets to when guaranteeing that intracavity atmospheric pressure keeps balance with the external world and promoting to use centrifugal force automatic injection solution, each reaction liquid can separate through the air, forms independent liquid section.

The rotary high-gradient magnetic field consists of a central shaft, a bearing, a (hollow) cylindrical magnet, two L-shaped rotating shafts, a shaft support and a fixing ring; wherein the diameter size of the central shaft (middle shaft) is equal to the inner diameter size of the bearing; the outer diameter of the bearing is equal to the inner diameter of the cylindrical magnet; the outer diameter of the cylindrical magnet is equal to the inner diameter of the circular microfluidic chip.

The central shaft comprises a middle shaft, an upper half shaft and a lower half shaft; the diameter size of the upper half shaft is equal to that of the lower half shaft but smaller than that of the middle shaft, and the length of the lower half shaft is larger than that of the upper half shaft.

The L-shaped rotating shaft is provided with an opening with the diameter equal to that of the upper half shaft and the lower half shaft at the position, close to the top, of the L-shaped long end, and the opening is used for connecting the central shaft and the rotating shaft; the tail part of the L-shaped short end is a semicircular contraction structure which is used for gathering magnetic induction lines, so that a vertical high-gradient magnetic field is generated at an air gap part between two L-shaped rotating shafts.

The shaft support is a shaft sleeve, the center of which is solid, and two sides of the shaft support are respectively provided with a shaft sleeve capable of accommodating the central shaft (lower half shaft) and a stepping motor shaft, and the shaft support is used for connecting a rotary high-gradient magnetic field and the stepping motor.

The fixing ring is a circular ring used for fixing the cylindrical magnet, so that the stepping motor can drive the L-shaped rotating shaft to rotate through the central shaft and the shaft support.

Preferably, the central shaft is made of photosensitive resin materials and is printed by a 3D printer.

Preferably, the bearing is of ferrous material and has a height of 2 mm.

Preferably, the cylindrical magnet is made of a rubidium-iron-boron material N52 strength material.

Preferably, the L-shaped rotating shaft is made of an iron material.

Preferably, the shaft support is made of photosensitive resin materials and is printed by a 3D printer.

Preferably, the fixing ring is made of photosensitive resin materials and is printed by a 3D printer.

In a second aspect, the invention provides an application of the rapid microorganism separation and detection platform in food-borne pathogenic bacteria detection.

According to the second aspect of the invention, a rapid microorganism separation and detection method based on catalytic color development of a nano nickel wire mesh and HRP-nanoflower is provided, the working principle of the method is that a blue signal substance is generated by color development of TMB catalyzed by the HRP-nanoflower, and quantitative detection of microorganisms is realized through mobile phone APP image analysis in combination with a circular microfluidic chip. Firstly, an immune nano nickel wire, a target microorganism, HRP-nanoflower, PBST cleaning solution and TMB solution are loaded in a spindle-shaped liquid storage cavity close to the center of a circle in a circular microfluidic chip in advance, the solution is transferred to a reaction cavity on the outer ring of the circular microfluidic chip through centrifugal force, and each reaction solution is separated through air to form an independent liquid segment. And then the nano nickel wire is moved by rotating a high gradient magnetic field through a stepping motor and reacts with the target microorganism and the HRP-nanoflower respectively to form a nickel wire-microorganism-nanoflower-HRP complex. Finally, the complex is washed and then transferred to a TMB liquid section, and TMB is catalyzed by HRP to generate a blue product (TMB oxide, TMB)_OX) And the mobile phone APP is used for carrying out gray level analysis, so that quantitative analysis (qualitative detection can also be realized) of the target microorganism is realized.

In the invention, the modification is carried out by streptavidin and biotin. Streptavidin has very high affinity with biotin, and 1 streptavidin can be combined with 4 biotin. By modifying streptavidin on the nickel wire and using biotinylated anti-target bacteria antibody as the antibody, the nickel wire can be linked to the antibody.

In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", "inner diameter", "outer diameter", etc., indicate orientations or positional relationships based on those shown in the drawings, and are used merely for convenience of description and simplification of description, but do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be considered as limiting the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

The compounds are abbreviated as follows:

EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;

NHSS: n-hydroxysuccinimide sulfonic acid sodium salt.

Example 1 construction of a microbial isolation assay System/platform

The system for separating and detecting microorganisms provided by the embodiment comprises a circular microfluidic chip 5, a rotating (high-gradient) magnetic field device 15, a connecting element, a stepping motor 16, a stepping motor driver 17, a fixing device 19, an embedded device (single chip microcomputer) 18 and a smart phone image analysis device 20.

The annular micro-fluidic chip is provided with six spindle-shaped liquid storage cavities 2 and arc-shaped reaction cavities 3 (the arc-shaped reaction cavities are arranged at the outer edge of the chip) connected with outlets of the liquid storage cavities, the liquid storage cavities are used for storing loaded liquid, and the arc-shaped reaction cavities are used for separating and detecting target microorganisms;

the rotating magnetic field device comprises two L-shaped rotating shafts 7 which can carry out circular motion under the driving of the stepping motor;

the fixing device is a three-layer bracket, the upper layer is detachably fixed with a circular micro-fluidic chip and a rotating magnetic field device which are in embedded connection, the middle layer is detachably fixed with a stepping motor, and the lower layer is detachably fixed with a stepping motor driver; the embedded connection annular micro-fluidic chip and the rotating magnetic field device are taken as a whole and are detachably connected with the stepping motor through the shaft support;

the structure of the circular micro-fluidic chip 5 is as follows:

the circular microfluidic chip is a copper coin structure with a circular hole in the center, and the diameter of the circular hole is larger than or equal to that of the cylindrical magnet 6 in the rotating magnetic field device, so that the circular microfluidic chip can smoothly penetrate through the cylindrical magnet;

the arc reaction cavity is separated by 2n air outlets 4 arranged on the annular microchannel to form n independent open arc cavities (used for forming independent liquid sections), and two air outlets are respectively arranged at two ends of the inner side of each cavity (namely the number of the liquid storage cavities is equal to the number of the arc cavities is equal to the number of the 1/2 air outlets);

6 spindle-shaped liquid storage cavities which are radially (uniformly) arranged at the center are arranged on the annular micro-fluidic chip, and the included angle between the liquid storage cavities is 60 degrees;

one end of each liquid storage cavity, which is close to the center of the chip, is provided with a sample inlet hole 1, the other end of each liquid storage cavity is provided with an outlet, and the outlet of each liquid storage cavity is communicated with an arc-shaped reaction cavity 3;

the rotating magnetic field device comprises a cylindrical magnet 6, two L-shaped rotating shafts 7, a central shaft 9, two bearings 11 and a fixing ring 14;

the central shaft is divided into an upper half shaft 12, a middle shaft and a lower half shaft 13; the length of the lower half shaft is greater than that of the upper half shaft; the upper half shaft fixes one L-shaped rotating shaft to one end of the cylindrical magnet through the opening formed in the tail part of the long end of the rotating shaft, and the lower half shaft fixes the other L-shaped rotating shaft to the other end of the cylindrical magnet through the opening formed in the tail part of the long end of the rotating shaft;

the annular micro-fluidic chip is nested in the middle of the cylindrical magnet, one L-shaped rotating shaft is positioned above the chip, the other L-shaped rotating shaft is positioned below the chip, an air gap is formed between the tail parts of the short ends of the two L-shaped rotating shafts, and the height of the air gap is greater than the thickness of the chip; a vertical (high gradient) magnetic field is generated at the air gap and correspondingly acts on an arc-shaped reaction cavity on the chip;

a lower half shaft 13 penetrating through the central shaft of the cylindrical magnet is connected with a stepping motor shaft through a shaft support 10;

the fixing ring 14 is used for fixing the cylindrical magnet, so that when the L-shaped rotating shaft rotates, the cylindrical magnet does not rotate, and the stepping motor 16 can drive the L-shaped rotating shaft to rotate through the central shaft and the shaft support.

The L-shaped rotating shafts are made of iron, and two strip-shaped small magnets 8 are further arranged at the tail part of the short end of each L-shaped rotating shaft to enhance the magnetic field.

The central shaft, the shaft support and the fixing ring are all made of non-magnetic materials or magnetic materials.

The system also includes an embedded device (single-chip microcomputer) 18 and smartphone image analysis equipment 20.

The single chip microcomputer is used for controlling the reaction time and the reaction speed.

The intelligent mobile phone image analysis equipment is used for detecting a reaction result. Preferably, the image analysis device is developed by using an android phone platform.

The material and specification are as follows:

the inner diameter of the annular micro-fluidic chip is 18mm, and the outer diameter of the annular micro-fluidic chip is 64 mm; the chip is provided with 6 liquid storage cavities with the same size and shape; the diameter of the liquid storage cavity sample inlet is 0.8 mm; the diameter of an air outlet arranged on the inner side of the annular microchannel is 0.6 mm;

the dimensions of the hollow cylindrical magnet (preferably N52) are: the outer diameter is 18mm, the inner diameter is 8mm, and the height is 28 mm;

the dimensions of the small bar magnet (preferably N52) are: 12.7mm × 6.4mm × 3.2 mm;

the bearing is made of iron; the size is as follows: the outer diameter is 8mm, the inner diameter is 5mm, and the height is 2 mm;

the shaft support is made of photosensitive resin and comprises an upper support shaft, a lower support shaft and a central solid part for connecting the upper support shaft and the lower support shaft, wherein the upper support shaft is used for embedding a lower half shaft of the shaft, and the lower support shaft is used for embedding a shaft of the stepping motor; the radius of the upper supporting shaft is 3mm, the length of the upper supporting shaft is 7mm, and the radius of the opening is 1.5 mm; the radius of the lower support shaft is 4mm, the length of the lower support shaft is 10mm, and the radius of the opening is 2.5 mm.

The structural schematic diagram of the circular microfluidic chip of the microorganism detection platform is shown in figure 1.

The overall structure schematic diagram of the microorganism detection platform is a rotating high gradient magnetic field and is shown in figure 2.

The structural diagram of the constituent parts is shown in FIG. 3.

The overall structure of the microorganism detection platform is schematically shown in FIG. 4.

A schematic process diagram of the principle of microbial detection is shown in FIG. 5.

A photograph of the constructed microorganism isolation detection system is shown in FIG. 8.

Example 2 method for rapidly separating and detecting food-borne pathogenic bacteria based on catalytic color development of nano nickel wire mesh and HRP-nanoflower

In this embodiment, with the food-borne pathogenic bacteria detection platform constructed in embodiment 1, first, a modified immune nano nickel wire solution, a sample solution to be detected containing a target microorganism, an HRP-nano flower solution, a cleaning solution 1(PBST solution), a cleaning solution 2(PBST solution), and a TMB color development solution (commercial TMB color development solution) are loaded in a spindle-shaped liquid storage cavity near the center of a circle in a circular microfluidic chip in advance, the solution is transferred to a reaction cavity on the outer ring of the circular microfluidic chip by centrifugal force, and each reaction solution is separated by air to form an independent liquid segment. And then the nano nickel wire is moved by rotating a high gradient magnetic field through a stepping motor and reacts with the target bacteria and the HRP-nanoflower respectively to form a nickel wire-bacteria-nanoflower-HRP complex. Finally, the complex is washed and moved to the liquid phase of a substrate (tetramethylbenzidine, TMB), and the blue product (TMB oxide, TMB) is generated by catalyzing the TMB with HRP_OX) And gray level analysis is carried out by utilizing the mobile phone APP, so that quantitative analysis of target bacteria is realized.

Taking the food-borne pathogenic bacteria, salmonella typhimurium, as an example, the specific method is as follows:

1. 1mL of Salmonella typhimurium (2.8X 10) cultured overnight (16h)⁹CFU/mL) was diluted with PBS buffer in a 10-fold gradient to give 2.8X 10³-2.8×10⁷CFU/mL of Salmonella culture samples.

2. Sequentially injecting 20 mu L of modified immune nano nickel wire solution, 20 mu L of salmonella samples with different concentrations, 20 mu L of immune nanoflower (HRP-nanoflower solution), two parts of 20 mu L of PBST cleaning solution and 20 mu L of TMB solution into a circular microfluidic chip sealed by 1% BSA; and then, injecting the liquid in the liquid storage cavity of the chip into an arc-shaped reaction cavity at the periphery of the chip by using the centrifugal force of 650-700 rpm for 3 s.

3. The chip is placed in a rotating high gradient magnetic field, and the stepping motor is started.

4. The rotating magnetic field firstly circulates for 2 times at the nano nickel line segment to form a nano nickel line network, and then the rotating magnetic field drives the nano nickel line network to enter the salmonella sample segment to circularly move for 50 times to capture bacteria to form magnetic bacteria; then, the magnetic field drives the magnetic bacteria to enter the immune nanoflower liquid section for circulating for 50 times to carry out bacteria marking to form nanoflower bacteria; performing 20 times of circular moving cleaning through two sections of PBST cleaning liquids respectively, and allowing the nanoflower bacteria to enter a TMB substrate section for 30 times of circular catalytic reaction; and finally, the rotating magnetic field drives the nanoflower bacteria reaching the catalytic reaction time to enter the original nano nickel wire liquid section, and the enzymatic reaction is finished.

5. And (3) carrying out image gray level analysis on the resultant by utilizing mobile phone image analysis software to obtain the bacterial concentration, uploading the result to a cloud platform for risk assessment, and giving an alarm when the bacterial concentration exceeds a set threshold value.

The preparation method of the modified immune nano nickel wire solution comprises the following steps:

(1) 1M Nickel chloride hexahydrate solution (99.9% pure) 75 μ L and ethylene glycol (99.8%) 15mL were mixed and added to a beaker and heated to 100 ℃;

(2) dropwise adding 0.5mL of hydrazine monohydrate to the mixture, and keeping the temperature at 100 ℃;

(3) heating was continued for 30min until a dark grey product formed and floated on the surface of the solution;

(5) dispersing the cleaned nano nickel wire in ethanol containing 0.03% w/v polyvinylpyrrolidone (PVP) to obtain 1mg/mL nano nickel wire solution;

(6) ultrasonically and uniformly mixing the nano nickel wire solution to form a uniform dispersion system; sucking 500 mu L of nano nickel wire solution by using a pipette gun, adding the nano nickel wire solution into a sample injection bottle, and cleaning the nano nickel wire solution for 2 times by using ultrapure water to remove a protective agent (ethanol) on the surface of the nickel wire;

(7) 100 mu L H₂O₂、100μL NH₄Adding OH and 500 mu L of deionized water into the cleaned nano nickel wire (carrying out hydroxylation modification on the nano nickel wire), and placing the mixed solution into a water bath at 80 ℃ for 30min to obtain the nano nickel wire modified with hydroxyl (the sample injection bottle is not covered);

(9) preparing 1% [3- (methoxylsilyl) propyl ] succinic anhydride solution (10 mu L of [3- (methoxylsilyl) propyl ] succinic anhydride +990 mu L of absolute ethanol) by using absolute ethanol, resuspending and drying the hydroxyl-modified nano nickel wire by using the 1% [3- (methoxylsilyl) propyl ] succinic anhydride solution (carrying out carboxylation modification on the nano nickel wire), and then placing the nano nickel wire on a blending instrument for overnight reaction at room temperature for 15h to obtain the carboxyl-modified nano nickel wire;

(10) the magnetic frame is used for recovering the nano nickel wire modified with carboxyl on the next day, and the nano nickel wire is washed for 3 times by absolute ethyl alcohol so as to remove the residual carboxylation reagent on the surface; cleaning with ultrapure water for 2 times to remove residual ethanol; then adding 500 mu L of 1M NaOH solution (pH 9, using HCl to adjust pH), reacting for more than 5h (visual observation shows that the water solubility of the nickel wire solution is good, namely the carboxyl is fully opened);

(11) washing the carboxyl-modified nano nickel wire soaked in the alkaline solution with ultrapure water for 3 times (to remove residual NaOH), and washing with a PB solution with pH of 7.4 for 1 time;

(13) washing the nano nickel wire modified with the activated carboxyl for 2 times by using a PB solution with pH 7.4 to remove residual EDC and NHSS;

(14) redissolving and modifying the nano nickel wire with the activated carboxyl by using 420 mu L of PB solution with pH 7.4, then adding 41.67 mu L (50 mu g) of 1.2mg/mL streptavidin solution and 2 mu L of 1% skim milk (used for binding the streptavidin and the nickel wire and stabilizing a reaction system), and reacting for 1h at room temperature;

(15) washing the nano nickel wire modified with the activated carboxyl by a PB solution with pH 7.4 for 2 times to remove unbound streptavidin; then adding 1% skim milk (dissolved by PB solution with pH 7.4) 500 μ L and sealing for 1h to obtain streptavidin nickel wire;

(16) adding 60 mu g of streptavidin nickel wire into 500 mu L of PBS solution, carrying out magnetic separation and cleaning for 2 times, and removing the skim milk solution;

(17) then re-dissolving the nano nickel wire by using 500 mu L of PBS solution, adding 4 mu g of anti-salmonella typhimurium biotinylated polyclonal antibody (2.25mg/mL), and incubating for 45min at room temperature; wherein the capture antibody specifically recognizes and binds to the target microorganism;

(18) and (5) carrying out magnetic separation and cleaning to remove redundant unbound antibodies, thus obtaining the antibody.

The preparation method of the HRP-nanoflower solution comprises the following steps:

(1) mu.g of anti-Salmonella typhimurium monoclonal antibody (anti-Salmonella typhimurium monoclonal antibody, purchased from Abcam, USA, at a concentration of 3.35mg/mL) and 180. mu.g of HRP enzyme (5mg/mL, 36. mu.L) were added to 1mL of PBS solution (3 mM);

(2) then 20. mu.L of 200mM CaCl₂Adding the solution (prepared by ultrapure water) into the mixed solution in the step (1), incubating at room temperature for 12h (self-assembly to form nanoflower), centrifuging at 15000rpm for 5min, collecting precipitate, washing with ultrapure water for 3 times, and centrifuging; after each redissolution, uniformly oscillating for 1min to obtain HRP-nanoflower material, redissolving with 400 μ L of ultrapure water, and storing at 4 ℃.

The graph of the gray value and the HRP-nanoflower with different concentrations obtained by the image analysis of the smartphone is shown in FIG. 6.

FIG. 7 is a calibration curve established for the detection of Salmonella typhimurium of the present invention, which can be expressed as G ═ 6.49 xln (C)_B)+200(R²0.99), where G is the gray value, G_BIs the salmonella concentration. The result shows that the gray value and the bacterial concentration have good linear relation, and the lower detection line can reach 56 CFU/mL.

Example 3

1. To verify blue TMB_OXAs feasibility of output signal, different concentrations of TMB of APP were analyzed using smartphone image developed in the group_OXAnd carrying out image acquisition and analysis. To obtain different concentrations of TMB_OXThe method comprises the steps of carrying out multiple-ratio dilution (1/2 original concentration dilution) on 500 mu M immune nanoflowers to obtain nanoflowers with different concentrations (2 mu M-500 mu M), then catalyzing TMB solutions with the same volume and different concentrations by using the same volume of nanoflowers, carrying out image acquisition by using APP to obtain gray values, and carrying out 3 parallel tests on each group. The results of the experiment are shown in FIG. 6, TMB_OXConcentration C of_NFAnd the gray value G has a good linear relation, and as the concentration of the nanoflower increases, the blue color deepens and the gray value decreases. The linear model can be expressed as G-27.51 xln (C)_NF)+216.89(R²0.99). The results show that with TMB_OXHas a good linear relationship with the gray value of the color filter (1), which shows that the TMB_OXMay be used as the output signal.

2. Parameter optimization

The automatic detection process of bacteria mainly comprises the following steps: separation, washing and catalysis. In order to obtain the best detection result, the variables involved in the experimental process need to be optimized. The nano nickel wire dosage, the motor running speed, the motor cycle times, the immune nanoflower dosage and the catalysis time have important influences on the bacteria separation efficiency and the signal output intensity, so that the invention carries out corresponding parameter optimization experiments.

(1) Nickel wire usage optimization

Usage amount of nano nickel wireThe separation efficiency of bacteria is influenced, so that the optimal using amount of the immune nickel wire is determined by the separation efficiency of various amounts of nano nickel wires for capturing salmonella typhimurium. Different amounts (20. mu.g, 40. mu.g, 60. mu.g, 80. mu.g and 100. mu.g) of immunonickel wires (1mg/mL) were injected into the circular microfluidic chip to capture 20. mu.L of immunonickel wires at a concentration of 4.8X 10, respectively⁶CFU/mL Salmonella typhimurium, the bacterial number is obtained by a plate counting method, and the separation efficiency of different amounts of immunonickel lines is calculated. When the amount of immunonickel wire used was increased from 20. mu.g to 60. mu.g, the separation efficiency increased from 71% to 90%. However, when the amount of the immunonickel wire used was increased to 80 μ g and 100 μ g, there was no significant change in the separation efficiency, indicating that 60 μ g of the immunonickel wire was sufficient for capturing the target bacteria. Therefore, the optimal dosage of the immunonickel line is selected to be 60 mug by the invention (figure 9).

(2) Motor rotation speed optimization

The rotation speed of the motor directly influences the time of the contact of the immune nickel wire and bacteria, thereby influencing the separation efficiency. 60 mu g of immune nano nickel wire and 5 multiplied by 10 are mixed⁶CFU/mL of Salmonella typhimurium reacted at different rates (4.8-12 degrees/s). When the rotating speed of the motor is reduced from 12 degrees/s to 6 degrees/s, the separation efficiency is increased from 73.8 percent to 91.4 percent, and when the rotating speed is continuously reduced to 4.8 degrees/s, the separation efficiency is slightly increased (the separation efficiency is slightly increased)<2.6%) so that the optimal motor speed of the present invention is 6 degrees/s (fig. 10).

(3) Motor cycle number optimization

The number of motor cycles also directly affects the time of the immunonickel wire in contact with the bacteria, thereby affecting the separation efficiency. 60 mu g of immune nano nickel wire and 7.2 multiplied by 10 are mixed⁶CFU/mL of Salmonella typhimurium were cycled at a rate of 20 s/cycle for various times. When the number of cycles was 30, the separation efficiency was 79%, when the number of increased cycles was 40, the separation efficiency was 86%, when the number of increased cycles was 50, the separation efficiency was increased to 90%, and when the number of cycles was 60, the separation efficiency was 92%, which remained almost unchanged, so that the optimum number of cycles of the present invention was 50 (fig. 11).

(4) Immune nanoflower dosage optimization

The amount of the immune nanoflower used may affect the intensity of the output signal and thus the sensitivity of the biosensor. Under otherwise identical conditions, different volumes of the immune nanoflower were used, each at a concentration of 2.8X 10. mu.L⁶Carrying out binding reaction on CFU/mL magnetobacteria to form a double-antibody sandwich complex; after being washed by PBST washing liquor, the complex catalyzes TMB to generate blue TMB_OXA product; and finally, carrying out gray level analysis by using the mobile phone APP. The grey value decreased from 116.32 to 96.29 when the volume of the immunized nanoflower increased from 5 μ g to 7.5 μ g. The volume of the immunized nanoflower was continued to increase to 10 μ g with a grey value of 93.74, slightly decreasing, however, when the volume of the immunized nanoflower was continued to increase to 12.5 μ g or 15 μ g, the grey values were 88.73, 83.45, respectively, with no significant change. This indicates that 7.5. mu.g of the immunized nanoflower is sufficient for catalytic color development. Therefore, the optimal amount of the selected immune nanoflower of the present invention was 7.5 μ g (fig. 12).

(5) Catalyst time optimization

The enzyme catalysis time is one of the important factors affecting the sensitivity of the biosensor, and it directly affects the concentration of the catalytic product. Therefore, the use concentration is 2.8X 10⁶And (3) respectively catalyzing TMB with the same volume for different circulation times by using a nickel wire-bacterium-nanoflower-HRP complex formed by reaction of CFU/mL target bacteria: 10. 20, 30, 40, 50. When the number of cycles was increased from 10 to 20, the grey value was decreased from 134.52 to 101.46, since the same amount of nickel wire-bacteria-nanoflower-HRP complex catalyzed more TMB for a longer catalytic time, resulting in more TMB_OX. The gray level value is slightly decreased to 95.95 by increasing the cycle number to 30, and the gray level values are substantially unchanged by continuing to increase the cycle numbers to 40 and 50 (93.14 and 92.05). The optimal number of cycles for the enzyme catalysis time is therefore 30 cycles (FIG. 13).

3. System performance evaluation

In order to evaluate the performances of the optical biosensor, such as sensitivity, specificity and the like, the invention uses the salmonella typhimurium as a research model, and develops a system performance evaluation experiment, including the detection of the salmonella typhimurium with different concentrations, the detection of non-target bacteria (staphylococcus aureus, vibrio parahaemolyticus and escherichia coli K12) and the detection of a labeled chicken sample.

(1) Salmonella culture sample detection

The invention takes the salmonella typhimurium as a detection model, and utilizes the biosensor to detect different concentrations (2.8 multiplied by 10) under the optimal condition³-2.8×10⁷CFU/mL) were tested to establish a mathematical model. Each sample concentration was subjected to 3 replicates and PBS buffer was used as a blank. When the concentration of Salmonella typhimurium is from 5.6X 10³CFU/mL increased to 5.6X 10⁷CFU/mL, TMB_OXThe color changed from colorless to dark blue. Measuring TMB through smartphone APP_OXTo perform image analysis to obtain the gray value thereof. The results show that the gray value G and the concentration C of the salmonella typhimurium_BHas good linear relation and can be expressed as G ═ 6.49 Xln (C)_B)+200(R²0.99), the lowest detection limit is 56CFU/mL by calculation, and the detection time is less than 1h (fig. 7).

(2) Specificity detection

The specificity of the optical biosensor mainly depends on a biotinylated polyclonal antibody resisting salmonella typhimurium and a monoclonal antibody resisting salmonella typhimurium. The invention is as follows 10⁶CFU/mL Salmonella typhimurium as the target bacterium, 10⁶CFU/mL of Staphylococcus aureus, Vibrio parahaemolyticus, and Escherichia coli K12 were used as non-target bacteria for the experiments. The grey values of the blank, staphylococcus aureus, vibrio parahaemolyticus and escherichia coli K12 were 152.7, 154.0, 148.6, 152.2, respectively, which were much higher than the grey value of the target bacteria of 105.4, indicating that the biosensor had good specificity (fig. 14).

(3) Detection of salmonella in labeled chicken samples

In order to verify the capability of the biosensor to detect real samples, the optical biosensor is used for carrying out Salmonella typhimurium (2.8 multiplied by 10) with different concentrations on the marked chicken samples³-2.8×10⁷CFU/mL). The detection result of the marked chicken sample is consistent with the detection result trend of the cultured sample, and the detection result trend can be calculatedTo obtain a concentration of 2.8X 10³-2.8×10⁷The recovery rate of the CFU/mL labeled chicken sample is 97.5% -101.8%, and the average recovery rate is 99.9%, which indicates that the optical biosensor can be used for detecting the salmonella typhimurium in the chicken sample (FIG. 15).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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