CN110734853B - Microorganism detection device and detection method - Google Patents
Microorganism detection device and detection method Download PDFInfo
- Publication number
- CN110734853B CN110734853B CN201911072020.9A CN201911072020A CN110734853B CN 110734853 B CN110734853 B CN 110734853B CN 201911072020 A CN201911072020 A CN 201911072020A CN 110734853 B CN110734853 B CN 110734853B
- Authority
- CN
- China
- Prior art keywords
- microorganism
- magnetic field
- substrate
- magnetic
- magnet body
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Abstract
The invention provides a microorganism detection device and a detection method. Firstly, generating a local high gradient magnetic field by using saw-tooth soft magnetic sheets arranged between cylindrical magnet magnetizing magnets which are mutually repulsive in pairs; capturing the magnetic beads modified by the biological recognition materials in a channel correspondingly arranged outside the magnetic field by using the high-gradient magnetic field to form a magnetic bead chain; then, capturing the target microorganisms flowing through the channel by using the magnetic bead chain to realize the separation of the target object and the background; then injecting another enzyme compound modified by biological recognition material to combine with the target microorganism; and finally, injecting a substrate corresponding to the enzyme, catalyzing the substrate by using the enzyme, and detecting a catalytic product to realize the determination of the target microorganism. A corresponding microorganism detection device is developed based on the detection method. By utilizing the microorganism detection device and the detection method provided by the invention, target microorganisms in a large-volume sample can be captured, separated and enriched, and the detection specificity and sensitivity are effectively improved.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a microbial detection device and a microbial detection method.
Background
The food safety problem caused by microbial contamination is seriously threatened to the health of human beings, so that the development of the screening of pathogenic microorganism contaminated food can effectively prevent the outbreak of food-borne diseases.
The traditional microorganism detection methods mainly comprise a culture method, a polymerase chain reaction method and an enzyme-linked immunosorbent assay method. The whole detection process of the culture method is longer; the enzyme-linked immunosorbent assay has short detection time, but has lower sensitivity and accuracy; although the polymerase chain reaction method has advantages of short detection time, high sensitivity, good specificity, etc., it requires a professional to perform complicated nucleic acid extraction. Therefore, the development of a novel pathogenic microorganism detection method has important research significance and application value.
Disclosure of Invention
The invention aims to provide a microorganism detection device.
Another object of the present invention is to provide a method for detecting microorganisms based on magnetic bead chains and enzymatic reactions.
In order to achieve the object, in a first aspect, the invention provides a microorganism detection device, which comprises a magnetic field unit, a microelectrode detection unit and a peristaltic pump, wherein the units are sequentially connected through a pipeline; wherein the peristaltic pump may also employ other fluid control devices.
The magnetic field unit is used for capturing target microorganisms and performing enzymatic reaction;
the microelectrode detection unit is used for detecting products of enzymatic reaction;
the peristaltic pump is used to regulate the flow or velocity of fluid.
The magnetic field unit consists of a magnet body and a pipeline wound outside the magnet body or surrounded outside the magnet body through a fixing part; one end of the pipeline is a sample inlet (the inlet end is connected with the sample supply device), and the other end of the pipeline is a sample outlet (the outlet end is connected with a sample inlet on the surface of the PDMS mould of the microelectrode detection unit); the position of the pipeline surrounding the outer side of the magnet body corresponds to the position of the annular soft magnetic sheet in the magnet body;
the magnet body consists of n cylindrical magnets which are sequentially stacked together, coaxial and mutually repulsive in pairs, n-1 annular soft magnetic sheets of which the outer edges are in a sawtooth shape and the outer diameters are the same as those of the cylindrical magnets, and a fixing device; the cylindrical magnets and the annular soft magnetic sheets are coaxial and are sequentially arranged in a crossed manner and are tightly fixed together through a fixing device; wherein n is an integer of 2 or more (preferably n ═ 6);
the microelectrode detection unit is formed by plasma bonding of a circular substrate with an Ag/AgCl reference electrode array and a cylindrical PDMS (polydimethylsiloxane) mould which is coaxial with the substrate and positioned above the substrate, and the outer diameter of the substrate is larger than or equal to that of the cylindrical PDMS mould;
the Ag/AgCl reference electrode array consists of a common electrode positioned at the center of the substrate, a plurality of single electrodes arranged along the circumferential direction of the substrate and a conductive material, wherein the common electrode and the single electrodes are respectively and independently connected with the conductive material; the common electrode and the single electrode are formed by dispensing conductive nano-silver ink on the surface of the substrate, which is in contact with the PDMS mold, and chlorinating ferric trichloride;
a plurality of channels which are radially arranged by taking the common electrode as the center are arranged on the surface of the PDMS mold, which is in contact with the substrate, one inlet end of each channel is connected with the single electrode, and one outlet end of each channel is connected with the common electrode; the inlet of each flow channel is communicated with a sample inlet which is arranged on the surface of the PDMS mold and is positioned right above the single electrode, and the outlet of each flow channel is communicated with a sample outlet which is arranged on the surface of the PDMS mold and is positioned right above the common electrode (the sample outlet is connected with one end of a peristaltic pump, and the other end of the peristaltic pump is connected with a waste liquid collecting container); the center of the PDMS mold is connected with a common electrode on the substrate through a conductive material.
Preferably, the fixing device of the magnet body is used for connecting the cylindrical magnet and the annular soft magnetic sheet in series and fixing the cylindrical magnet and the annular soft magnetic sheet through the close fit of a rod with threads at two ends and a nut; the rod is made of a material that is non-magnetic and has a certain hardness.
Preferably, the magnetic field unit consists of a magnet body and a pipeline surrounding the outside of the magnet body through a fixing part; the fixing part is a photosensitive resin mold, the photosensitive resin mold is designed according to the shape of the magnet body, grooves are formed in the surface of the photosensitive resin mold and used for fixing a pipeline which is wound on the outer side of the magnet body.
Preferably, the substrate is an insulating polymer, glass, silicon, or the like. More preferably a glass substrate.
Preferably, the tubing is teflon tubing.
Preferably, the bar is an aluminium bar. The two ends of the aluminum bar are cylindrical and are provided with threads.
Preferably, the photosensitive resin mold is printed by a 3D printer.
Preferably, the runner on the PDMS mold is made by photolithography and/or etching.
Furthermore, the cylindrical magnet has an outer diameter of 20-60 mm (preferably 40mm), an inner diameter of about 5mm, and a thickness of about 5 mm.
Furthermore, the size of the outer diameter and the inner diameter of the annular soft magnetic sheet with the sawtooth-shaped outer edge is matched with the size of the outer diameter and the inner diameter of the cylindrical magnet, and the thickness is about 2 mm.
Further, the outer diameter of the pipeline is matched with the thickness of the annular soft magnetic sheet.
Further, the outer diameter of the rod is sized to match the inner diameter of the cylindrical magnet.
Furthermore, the outer diameter of the substrate with the Ag/AgCl reference electrode is about 40 mm.
Further, the outer diameter of the PDMS mold is 39mm, and the height thereof is about 5 mm.
Furthermore, the public electrode is circular, and the external diameter is about 5 mm.
Further, the single electrode has a square shape and has a size of (2 to 8mm) × (2 to 8mm), preferably 2mm × 8 mm.
Further, the included angle between the runners on the PDMS mold is 360 °/m, where m is the number of runners (preferably, m is 6).
The length of the flow channel is matched with the linear distance from the common electrode to the single electrode, the width of the flow channel is matched with the outer diameter size of a sample inlet or a sample outlet on the surface of the PDMS mold, the depth of the flow channel is about 1mm, and the number of the flow channels is consistent with the number of the single electrodes.
Further, the photosensitive resin mold has a thickness greater than an outer diameter of the pipe.
Further, the size of the injection port on the PDMS mold surface matches the outer diameter of the tubing.
According to the detection device, the peristaltic pump is used for pumping a microorganism sample to flow through the pipeline, the microorganism in the sample is captured in the pipeline by the magnetic beads, then other samples are pumped in through the micro valve, and other impurities in the sample flow out of the pipeline and are recycled into the waste liquid collection container.
Further, the peristaltic pump can be connected with a microprocessor for controlling the liquid flow or flow rate.
Furthermore, the cylindrical magnet and the annular soft magnetic sheet with the sawtooth-shaped outer edge are provided with coaxial calibrators which are formed by tightly matching a threaded aluminum rod and a nut, so that the tips of the gears can form a chain-shaped effect on magnetic beads in the channel.
Further, the length of the coaxial calibrator is preferably 65mm, and the outer diameter is preferably 5 mm. The nut is an M5 ingot type nut.
When the magnetic field unit is manufactured, firstly, two cylindrical magnets and zigzag soft magnetic sheets which are mutually exclusive are sequentially arranged in a crossed manner, connected by an aluminum bar, and screwed tightly by nuts at two ends; then selecting a Teflon tube with a proper length to be wound on the photosensitive resin mold according to the track of the groove; and finally combining them.
In a second aspect, the invention provides the use of the detection device for the detection of microorganisms.
In a third aspect, the invention provides a microorganism detection method based on magnetic bead chains and enzymatic reaction, which comprises the steps of firstly utilizing zigzag soft magnetic sheets arranged between cylindrical magnet magnetizing magnets which repel each other in pairs to generate a local high gradient magnetic field; capturing the magnetic beads modified by the biological recognition materials in a channel correspondingly arranged outside the magnetic field by using the high-gradient magnetic field to form a magnetic bead chain; then the magnetic bead chain is used for capturing the target microorganism flowing through the channel, so that the separation of the target object and the background is realized; then injecting another enzyme compound modified by biological recognition material to combine with the target microorganism; and finally, injecting a substrate corresponding to the enzyme, catalyzing the substrate by using the enzyme, and detecting a catalytic product by using a linear scanning voltammetry method through an array printing film Ag/AgCl reference electrode to detect the concentration of the microorganism.
The microorganism detection method comprises the following steps:
1) injecting the magnetic beads modified by the biological recognition materials into a magnetic field unit of the microorganism detection device, and capturing the magnetic beads modified by the biological recognition materials in a corresponding pipeline outside the magnetic field by using the high-gradient magnetic field to form a magnetic bead chain;
2) injecting a microorganism sample into the magnetic field unit, and capturing target microorganisms in the sample by using a magnetic bead chain;
3) then injecting an enzyme complex modified with another biological recognition material into the magnetic field unit, wherein the enzyme complex is combined with the target microorganism captured on the magnetic bead chains;
4) finally, injecting a substrate corresponding to the enzyme into the magnetic field unit, after the enzymatic reaction is finished, enabling the product to pass through the microelectrode detection unit and be connected with an electrochemical workstation for linear voltammetry scanning to obtain resistance values corresponding to microorganism samples with different concentrations, and establishing a standard curve between the microorganism concentration and the resistance values; and calculating to obtain the concentration corresponding to the resistance value of the microbial sample to be detected according to the established standard curve.
The magnetic beads modified by the biological recognition material can be magnetized by an external magnetic field, and a magnetic bead chain is formed in the channel, so that the magnetic beads can be effectively prevented from being gathered, and the magnetic beads are uniformly captured in the pipeline (Teflon pipe).
Preferably, the biological recognition material is a monoclonal antibody or polyclonal antibody capable of specifically recognizing the target microorganism.
Further, the preparation method of the magnetic bead modified by the biological recognition material comprises the following steps: and mixing and incubating the magnetic beads modified by streptavidin with biotinylated antibodies. For example, streptavidin-modified magnetic beads and biotinylated monoclonal antibodies are mixed in a mixer for 45 minutes, washed to remove unbound materials, and then reconstituted with phosphate buffer to obtain monoclonal antibody-modified magnetic beads.
Further, the preparation method of the enzyme complex modified by the biological recognition material comprises the following steps: preparing a colloidal gold solution, dropwise adding the non-biotinylated antibody into the colloidal gold solution under the condition of stirring, and then adding enzyme for mixing. For example, a colloidal gold solution is taken in a small beaker and placed on a magnetic stirrer, the non-biotinylated polyclonal antibody is dropwise added into the colloidal gold solution, then the enzyme is added, the stirring is carried out for 1 hour, finally, the unbound polyclonal antibody and the enzyme are removed by centrifugation, and the polyclonal antibody-enzyme complex is obtained by redissolving with ultrapure water.
Further, a resistance value corresponding to the known concentration is obtained by linear sweep voltammetry, and a calibration curve between the concentration and the resistance value can be established by linear fitting:
y＝a×ln(x)+b
wherein a and b are constants, y is a resistance value, and x is a microorganism concentration.
In the present invention, the microorganism includes food-borne pathogenic bacteria (bacteria), preferably salmonella, more preferably salmonella typhimurium.
When the microorganism is salmonella typhimurium, the enzyme may be urease and the substrate may be urea. The monoclonal antibody can be anti-salmonella typhimurium monoclonal antibody, and the polyclonal antibody can be anti-salmonella typhimurium polyclonal antibody.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
by utilizing the microorganism detection device and the detection method provided by the invention, the magnetic bead chains formed in the high gradient magnetic field area can capture, separate and enrich target microorganisms in a large-volume sample through the construction of the magnetic field unit, and the detection sensitivity is effectively improved through the detection unit prepared by combining the array printing film Ag/AgCl reference electrode and the PDMS mold.
Drawings
Fig. 1 is a schematic structural view of a magnet body of a microorganism detection apparatus in embodiment 1 of the present invention.
FIG. 2 is a schematic view showing the overall structure of a magnetic field unit of the microorganism detection apparatus in example 1 of the present invention.
FIG. 3 is a schematic view showing the configuration of a microelectrode detecting unit of the microorganism detecting apparatus in example 1 of the present invention.
FIG. 4 is a schematic view showing the detection principle of the magnetic field unit of the microorganism detection apparatus in example 1 of the present invention.
FIG. 5 is a schematic view showing a detection scheme of microorganisms based on magnetic bead chains and enzymatic reactions in example 1 of the present invention.
FIG. 6 is a cross-sectional view of a magnetic field unit of the microorganism detection apparatus in example 1 of the present invention.
FIG. 7 is a schematic diagram of a process for preparing an array printed thin film Ag/AgCl reference electrode of the microorganism detection device in example 1 of the present invention.
FIG. 8 is a top view of an array printed thin film Ag/AgCl reference electrode of the microbial detection device of example 1 of the present invention.
FIG. 9 is a calibration curve between the resistance value and the microbial concentration obtained by the linear sweep voltammetry in example 2 of the present invention.
Fig. 10 is a graph showing the results of the capturing rate in the case where the magnetic field devices of example 2 of the present invention are composed of mutually repelling cylindrical magnets, mutually attracting cylindrical magnets and soft magnetic sheets without saw-tooth shapes, respectively.
FIG. 11 is a result of evaluating the stability of the microorganism detection apparatus in example 2 of the present invention.
In fig. 1 to 8: the device comprises a common electrode 1, a single electrode 2, a glass substrate 3, a PDMS mold 4, a sample inlet 5, a sample outlet 6, a flow channel 7, conductive materials 8 and 9, a cylindrical magnet 10, a sawtooth-shaped annular soft magnetic sheet 11, an aluminum rod 12, a nut 13, a photosensitive resin mold 14, a groove 15, a sample inlet 16, a sample outlet 17, a micro valve 18, a sample solution supply container 19, a sample solution supply container 20-Ag, 21-AgCl, a Teflon tube 22, a magnetic bead 23, a peristaltic pump 24, a waste liquid collection container 25, a multi-inlet single-outlet channel 26 and a magnetic field device 27.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products. The magnetic beads used had a particle size of 150nm and were obtained from the American nanotechnology company, model MHS-150-10.
In the description of the present invention, unless otherwise specified, the terms "upper", "lower", "inside", "outside", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
EXAMPLE 1 microorganism detection apparatus
The microorganism detection device provided by the embodiment comprises a magnetic field unit, a microelectrode detection unit and a peristaltic pump, wherein the units are sequentially connected through a Teflon tube.
Wherein the magnetic field unit is used for capturing target microorganisms and performing enzymatic reaction; the microelectrode detection unit is used for detecting products of enzymatic reaction; peristaltic pumps are used to regulate the flow or velocity of fluid.
The magnetic field unit consists of a magnet body and a Teflon tube which surrounds the outside of the magnet body through a fixing part; one end of the Teflon tube is a sample inlet (the inlet end is connected with the sample supply device), and the other end is a sample outlet (the outlet end is connected with a sample inlet on the surface of the PDMS die of the microelectrode detection unit); the position of the Teflon tube surrounding the outer side of the magnet body corresponds to the position of the annular soft magnetic sheet in the magnet body.
The fixing part is a photosensitive resin mold, the photosensitive resin mold is designed according to the shape of the magnet body, grooves are formed in the surface of the photosensitive resin mold and used for fixing a pipeline which is wound on the outer side of the magnet body.
In the magnetic field unit, a magnet body (figure 1) comprises a plurality of three pairs of cylindrical magnets 10 which are sequentially stacked together, coaxial and mutually repulsive in pairs, and five annular soft magnetic sheets 11 with saw-toothed outer edges; the cylindrical magnet and the annular soft magnetic sheet have the same outer diameter and are fixed through the close fit of a threaded aluminum bar 12 and nuts 13 on two sides; the cylindrical magnets and the annular soft magnetic sheets which are mutually repulsive in pairs are sequentially arranged in a crossed manner.
The photosensitive resin mold 14 is designed according to the shape of the magnet body, a groove 15 is formed in the surface of the photosensitive resin mold and used for fixing a Teflon tube which is wound on the outer side of the magnet body in a fixing mode, one end of the Teflon tube is a sample inlet 16, the other end of the Teflon tube is a sample outlet 17, the position, around the outer side of the magnet body, of the Teflon tube corresponds to the position of the annular soft magnetic sheet, and the overall structure schematic diagram of the magnetic field unit is shown in figure 2.
The microelectrode detection unit (figure 3) is formed by plasma bonding of a round glass substrate with an Ag/AgCl reference electrode array and a PDMS mold. The Ag/AgCl reference electrode array is formed by arranging a central common electrode 1 and six square electrodes 2 (single electrodes) in the circumferential direction. Further, the glass substrate 3 is dispensed with the conductive nano silver ink (plated with silver), and then chlorinated by ferric trichloride to finally form the glass substrate with the Ag/AgCl reference electrode array. The closed channel (flow channel) formed by the PDMS mold 4 and the glass substrate is used for storing and circulating a detection sample, namely, the sample flows in through the inlet 5 (sample inlet) and flows out through the outlet 6 (sample outlet), flows through the reference electrode through the cuboid-shaped flow channel 7, and is finally connected to an electrochemical workstation through conductive materials (graphene conductive adhesive) 8 and 9 to perform linear voltammetry scanning.
In the microorganism detection apparatus based on the magnetic bead chain and the enzymatic reaction of the present invention, the detection principle of the magnetic field unit for capturing the target microorganism and performing the enzymatic reaction (fig. 4) is as follows:
s1, fixing cylindrical magnets and annular soft magnetic sheets (saw-toothed soft magnetic sheets) with saw-toothed outer edges which are arranged in sequence in a crossed mode by means of close matching of aluminum bars with threads on two sides and nuts to construct a magnetic field device (magnet body);
s2, designing a smooth groove channel made of photosensitive resin materials according to the shape of the magnetic field device to fix the Teflon tube, and nesting the smooth groove channel outside the magnetic field device;
s3, pumping magnetic beads modified by biological recognition materials through a peristaltic pump, and enabling the magnetic beads to form a magnetic bead chain in the Teflon tube under a local high-gradient magnetic field;
s4, pumping a microorganism sample, and capturing the microorganism by using magnetic beads;
and S5, controlling the pumping of other samples through the micro valve, and finally pumping out the catalytic product for detection.
As shown in fig. 5 to 8, a microorganism testing apparatus includes a sample solution supply container 19 controlled by a micro valve 18, through which different sample solutions are sequentially supplied through a multi-inlet single-outlet channel 26; the magnetic field device 27 and the PDMS mold 4 are connected through a connecting pipe; the magnetic field device 27 comprises cylindrical magnets 10 and saw-tooth-shaped soft magnetic sheets 11 which are arranged in sequence in a crossed mode, aluminum bars 12 with threads on two sides are tightly matched with nuts 13, and a photosensitive resin mold 14 with an annular groove is additionally arranged to fix the connected Teflon tube 22; the teflon tube connects the entire device liquid flow.
The magnetic beads 23 are magnetized by a magnetic field device, and a magnetic bead chain is formed in the Teflon tube.
The outer diameter of the cylindrical magnet is 40mm, the inner diameter is 5mm, and the thickness is 5 mm.
The outer diameter of the sawtooth-shaped soft magnetic sheet is 40mm, the inner diameter of the sawtooth-shaped soft magnetic sheet is 5mm, and the thickness of the sawtooth-shaped soft magnetic sheet is 2 mm.
The length of the aluminum bar is preferably 65mm, and the outer diameter is preferably 5 mm. The nut is an M5 ingot type nut.
The photosensitive resin mold is formed by printing through a 3D printer.
The outer diameter of the Teflon tube is 2.0mm, and the inner diameter of the Teflon tube is 1.2 mm.
The multi-inlet single-outlet channel, the magnetic field device, the PDMS mold and the peristaltic pump 24 are all connected by Teflon pipes.
The peristaltic pump may also employ other fluid control devices.
The other end of the peristaltic pump is also connected with a waste liquid collecting container 25.
The array printing film Ag/AgCl reference electrode detection device (microelectrode detection unit) is manufactured by carrying out plasma bonding on a PDMS mould and a glass substrate with the array printing film Ag/AgCl (a round substrate with an Ag/AgCl reference electrode array).
And (3) dispensing the conductive nano-silver ink on a glass substrate, curing for 10 minutes in an air environment at 100 ℃ to form a silver electrode array, and chlorinating by ferric trichloride to obtain the conductive nano-silver ink.
The sheet size of each silver electrode (single electrode) was 2mm × 8mm, and the included angle was 60 degrees.
The outer diameter of the central common silver electrode is 5 mm.
Example 2 detection of microorganisms based on magnetic bead chains and enzymatic reactions
This example provides a method for detecting microorganisms based on magnetic bead chains and enzymatic reactions. According to the microorganism detection device of the embodiment 1, firstly, a local high gradient magnetic field is generated by using the zigzag soft magnetic sheets arranged between two cylindrical magnet magnetizing magnets which are mutually repulsive; capturing the magnetic beads modified by the biological recognition materials in a channel correspondingly arranged outside the magnetic field by using the high-gradient magnetic field to form a magnetic bead chain; then the magnetic bead chain is used for capturing the target microorganism flowing through the channel, so that the separation of the target object and the background is realized; then injecting another enzyme compound modified by biological recognition material to combine with the target microorganism; and finally, injecting a substrate corresponding to the enzyme, catalyzing the substrate by using the enzyme, and detecting the microbial concentration by performing linear scanning voltammetry on a catalytic product through an array printing film Ag/AgCl reference electrode.
The specific method comprises the following steps:
1.1 mL of Salmonella typhimurium (concentration 1.1X 10) cultured overnight9CFU/mL), were diluted 10-fold in sequence with sterile PBS (pH 7.4, 10mM) to give a concentration range of 1.2X 106CFU/mL to 1.2X 101CFU/mL of bacterial sample.
2. And sequentially pumping 25 mu L of magnetic beads modified by monoclonal antibodies (anti-salmonella typhimurium monoclonal antibodies), 5mL of bacterial samples with different concentrations and 1mL of polyclonal anti-urease composite material into a Teflon tube channel outside the magnetic field device to obtain an enzyme-bacteria composite, capturing the enzyme-bacteria composite on the magnetic bead chain, and directly pumping the unbound waste back to a waste liquid collecting container.
3. And pumping 1mL of urea into the channel for catalysis, pumping a product into an array printing film Ag/AgCl reference electrode detection device after the catalysis is finished, and carrying out linear scanning voltammetry for detection to obtain a corresponding resistance value.
4. And performing linear fitting by using Excel software to obtain a calibration curve between the resistance value and the bacterial concentration.
The monoclonal antibody can be anti-salmonella typhimurium monoclonal antibody, and the polyclonal antibody can be anti-salmonella typhimurium polyclonal antibody.
The preparation method of the monoclonal antibody modified magnetic bead comprises the following steps: and mixing 20 mu L of magnetic beads modified by 1mg/mL streptavidin with 4 mu L of biotinylated monoclonal antibody in a mixing machine for 45 minutes, washing to remove unbound materials, and re-dissolving with 50 mu L of phosphate buffer solution to obtain the magnetic beads modified by the monoclonal antibody.
The preparation method of the multi-resistant-urease composite material comprises the following steps: putting the colloidal gold solution in a small beaker, placing the beaker on a magnetic stirrer, dropwise adding 3 mu L of non-biotinylated polyclonal antibody of 2.5mg/mL into the colloidal gold solution, then adding 120 mu g of urease, stirring for 1 hour, finally removing the unbound polyclonal antibody and urease through centrifugation, and re-dissolving with 100 mu L of ultrapure water to obtain the polyclonal antibody-urease composite material.
FIG. 9 is a calibration curve established for the detection of Salmonella typhimurium of the present invention, which can be expressed as:
y＝-0.048ln(x)+1.763 (R2＝0.987)
wherein y is a resistance value and x is a microbial concentration.
The result shows that the resistance value has a good linear relation with the bacterial concentration, and the lower detection limit can reach 20 CFU/mL.
Fig. 10 is a graph comparing the results of the trapping rate of the magnetic field apparatus of the present invention consisting of the mutually repelling cylindrical magnets, the mutually attracting cylindrical magnets and the saw-tooth shaped soft magnetic flakes. It can be seen that the capture efficiency of the magnetic field device of the present invention is significantly higher than that of the conventional soft magnetic sheet structure without zigzag for the same bacterial sample, which indicates that the detection device of the present invention plays an important role in capturing microorganisms with larger volume.
FIG. 11 is a stability test of the Ag/AgCl reference electrode detection device with array printing film according to the present invention, and the results show that different resistance values are obtained by performing linear sweep voltammetry detection on ammonium carbonate with different concentrations (ammonium carbonate is generated by urease catalysis substrate urea), the ammonium carbonate with different concentrations and the corresponding resistance values have good linear relationship, and y is-0.642 ln (x) +0.372 (R is R is-0.642 ln), (x is-0.372)20.981), further illustrating the greater stability of the reference electrode of the present invention in detection.
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.
Claims (10)
1. Method for the detection of microorganisms based on magnetic bead chains and enzymatic reactions, for non-disease diagnostic and therapeutic purposes, characterized in that it comprises the following steps:
1) injecting the magnetic beads modified by the biological recognition materials into a magnetic field unit of the microorganism detection device, and capturing the magnetic beads modified by the biological recognition materials in a corresponding pipeline outside the magnetic field by using a high-gradient magnetic field to form a magnetic bead chain;
2) injecting a microorganism sample into the magnetic field unit, and capturing target microorganisms in the sample by using a magnetic bead chain;
3) then injecting an enzyme complex modified with another biological recognition material into the magnetic field unit, wherein the enzyme complex is combined with the target microorganism captured on the magnetic bead chains;
4) finally, injecting a substrate corresponding to the enzyme into the magnetic field unit, after the enzymatic reaction is finished, enabling the product to pass through the microelectrode detection unit and be connected with an electrochemical workstation for linear voltammetry scanning to obtain resistance values corresponding to microorganism samples with different concentrations, and establishing a standard curve between the microorganism concentration and the resistance values; calculating to obtain the concentration corresponding to the resistance value of the microbial sample to be detected according to the established standard curve;
the enzyme is urease, and the substrate is urea; the microorganism detection device comprises a magnetic field unit, a microelectrode detection unit and a peristaltic pump, and all the units are connected in sequence through pipelines;
the magnetic field unit is used for capturing target microorganisms and performing enzymatic reaction;
the microelectrode detection unit is used for detecting products of enzymatic reaction;
the peristaltic pump is used for adjusting the flow or flow rate of the fluid;
the magnetic field unit consists of a magnet body and a pipeline wound outside the magnet body or surrounded outside the magnet body through a fixing part; one end of the pipeline is a sample inlet, and the other end of the pipeline is a sample outlet; the position of the pipeline surrounding the outer side of the magnet body corresponds to the position of the annular soft magnetic sheet in the magnet body;
the magnet body consists of n cylindrical magnets which are sequentially stacked together, coaxial and mutually repulsive in pairs, n-1 annular soft magnetic sheets of which the outer edges are in a sawtooth shape and the outer diameters of which are the same as those of the cylindrical magnets, and a fixing device; the cylindrical magnets and the annular soft magnetic sheets are coaxial and are sequentially arranged in a crossed manner and are tightly fixed together through a fixing device; wherein n is an integer greater than or equal to 2;
the microelectrode detection unit is formed by plasma bonding of a circular substrate with an Ag/AgCl reference electrode array and a cylindrical PDMS (polydimethylsiloxane) mold which is coaxial with the substrate and positioned above the substrate, and the outer diameter of the substrate is larger than or equal to that of the cylindrical PDMS mold;
the Ag/AgCl reference electrode array consists of a common electrode positioned at the center of the substrate, a plurality of single electrodes arranged along the circumferential direction of the substrate and a conductive material, wherein the common electrode and the single electrodes are respectively and independently connected with the conductive material; the common electrode and the single electrode are formed by dispensing conductive nano-silver ink on the surface of the substrate, which is in contact with the PDMS mold, and chlorinating ferric trichloride;
a plurality of channels which are radially arranged by taking the common electrode as the center are arranged on the surface of the PDMS mold, which is in contact with the substrate, one inlet end of each channel is connected with the single electrode, and one outlet end of each channel is connected with the common electrode; the inlet of each flow channel is communicated with a sample inlet which is arranged on the surface of the PDMS mold and is positioned right above the single electrode, and the outlet of each flow channel is communicated with a sample outlet which is arranged on the surface of the PDMS mold and is positioned right above the common electrode; the center of the PDMS mold is connected with a common electrode on the substrate through a conductive material.
2. The method of claim 1, wherein the fixing means of the magnet body is a cylindrical magnet and a ring-shaped soft magnetic thin plate connected in series and fixed by a tight fit of a bar and a nut with threads at both ends; the rod is made of a material that is non-magnetic and has a certain hardness.
3. The method of claim 2, wherein the magnetic field unit is composed of a magnet body and a pipe surrounding the outside of the magnet body through a fixing member; the fixing part is a photosensitive resin mold, the photosensitive resin mold is designed according to the shape of the magnet body, grooves are formed in the surface of the photosensitive resin mold and used for fixing a pipeline which is wound on the outer side of the magnet body.
4. The method of claim 3, wherein the substrate is an insulating polymer, glass, or silicon; and/or
The pipeline is a Teflon pipe; and/or
The bar is an aluminum bar; and/or
The photosensitive resin mold is formed by printing through a 3D printer; and/or
The runner on the PDMS mould is made by adopting a photoetching and/or etching method.
5. The method according to claim 3, wherein the cylindrical magnet has an outer diameter of 20 to 60mm, an inner diameter of 5mm, and a thickness of 5 mm; and/or
The outer diameter and the inner diameter of the annular soft magnetic sheet with the sawtooth-shaped outer edge are matched with those of the cylindrical magnet, and the thickness of the annular soft magnetic sheet is 2 mm; and/or
The outer diameter of the pipeline is matched with the thickness of the annular soft magnetic sheet; and/or
The outer diameter of the rod is matched with the inner diameter of the cylindrical magnet; and/or
The outer diameter of the substrate with the Ag/AgCl reference electrode is 40 mm; and/or
The outer diameter of the PDMS mold is 39mm, and the height of the PDMS mold is 5 mm; and/or
The common electrode is circular, and the outer diameter of the common electrode is 5 mm; and/or
The single electrode is square, and the size is (2-8 mm) × (2-8 mm); and/or
The included angle between each runner on the PDMS mold is 360 degrees/m, and m is the number of runners; and/or
The photosensitive resin mold has a thickness greater than an outer diameter of the pipe.
6. The method according to claim 1, wherein the biological recognition material is a monoclonal antibody or polyclonal antibody that can specifically recognize the target microorganism.
7. The method of claim 1, wherein the method for preparing the magnetic beads modified with the biological recognition material comprises: mixing and incubating magnetic beads modified by streptavidin with biotinylated antibodies; and/or
The preparation method of the enzyme complex modified by the biological recognition material comprises the following steps: preparing a colloidal gold solution, dropwise adding the non-biotinylated antibody into the colloidal gold solution under the condition of stirring, and then adding enzyme for mixing.
8. The method of any one of claims 1 to 7, wherein the microorganisms comprise food-borne pathogenic bacteria.
9. The method of claim 8, wherein the microorganism is Salmonella.
10. The method of claim 9, wherein the microorganism is salmonella typhimurium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911072020.9A CN110734853B (en) | 2019-11-05 | 2019-11-05 | Microorganism detection device and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911072020.9A CN110734853B (en) | 2019-11-05 | 2019-11-05 | Microorganism detection device and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110734853A CN110734853A (en) | 2020-01-31 |
| CN110734853B true CN110734853B (en) | 2021-07-30 |
Family
ID=69272233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911072020.9A Active CN110734853B (en) | 2019-11-05 | 2019-11-05 | Microorganism detection device and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110734853B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112362711B (en) * | 2020-11-11 | 2021-08-17 | 重庆大学 | Microorganism detection device and detection method |
| CN113588957B (en) * | 2021-07-16 | 2023-06-30 | 中国农业大学 | Microorganism separation detection system and detection method |
| CN113721015B (en) * | 2021-09-08 | 2023-03-21 | 中国农业大学 | Automatic microorganism detection device, system and method |
| CN113817602B (en) * | 2021-09-18 | 2024-04-05 | 中国农业大学 | Simple microorganism separation device and separation method |
| CN114250148B (en) * | 2021-12-30 | 2024-04-02 | 杭州电子科技大学 | 3D immune magnetic grid separation device, system and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101865912A (en) * | 2009-04-14 | 2010-10-20 | 南京大学 | Fast chemiluminescence immune detection system and analysis method |
| CN102002461A (en) * | 2010-10-14 | 2011-04-06 | 中国人民解放军军事医学科学院卫生装备研究所 | IMB (immunomagnetic bead) based sequential fluid type fully-automatic magnetic separation device and method |
| CN103424447A (en) * | 2013-08-22 | 2013-12-04 | 中国科学院上海微系统与信息技术研究所 | Nano-particle enhancement detection device based on non-modified monolayer graphene being used as working electrode and application thereof |
| WO2014060454A1 (en) * | 2012-10-16 | 2014-04-24 | Mode Diagnostics Limited | Immunoassay using electrochemical detection |
| CN104833802A (en) * | 2015-04-24 | 2015-08-12 | 中国农业大学 | Microbial detection method based on immunomagnetic separation and urease catalysis |
| CN107202880A (en) * | 2017-05-24 | 2017-09-26 | 中国农业大学 | A kind of microorganism detection method analyzed based on electrochemical impedance phase angle |
| CN108287236A (en) * | 2017-11-24 | 2018-07-17 | 中国农业大学 | A kind of biological sensor and its application based on High-gradient Magnetic separation and quantum dot |
-
2019
- 2019-11-05 CN CN201911072020.9A patent/CN110734853B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101865912A (en) * | 2009-04-14 | 2010-10-20 | 南京大学 | Fast chemiluminescence immune detection system and analysis method |
| CN102002461A (en) * | 2010-10-14 | 2011-04-06 | 中国人民解放军军事医学科学院卫生装备研究所 | IMB (immunomagnetic bead) based sequential fluid type fully-automatic magnetic separation device and method |
| WO2014060454A1 (en) * | 2012-10-16 | 2014-04-24 | Mode Diagnostics Limited | Immunoassay using electrochemical detection |
| CN103424447A (en) * | 2013-08-22 | 2013-12-04 | 中国科学院上海微系统与信息技术研究所 | Nano-particle enhancement detection device based on non-modified monolayer graphene being used as working electrode and application thereof |
| CN104833802A (en) * | 2015-04-24 | 2015-08-12 | 中国农业大学 | Microbial detection method based on immunomagnetic separation and urease catalysis |
| CN107202880A (en) * | 2017-05-24 | 2017-09-26 | 中国农业大学 | A kind of microorganism detection method analyzed based on electrochemical impedance phase angle |
| CN108287236A (en) * | 2017-11-24 | 2018-07-17 | 中国农业大学 | A kind of biological sensor and its application based on High-gradient Magnetic separation and quantum dot |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110734853A (en) | 2020-01-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110734853B (en) | Microorganism detection device and detection method | |
| Zhang et al. | Detection of pathogenic microorganisms by microfluidics based analytical methods | |
| Park et al. | 3D-printed microfluidic magnetic preconcentrator for the detection of bacterial pathogen using an ATP luminometer and antibody-conjugated magnetic nanoparticles | |
| Frenea-Robin et al. | Basic principles and recent advances in magnetic cell separation | |
| US20120115167A1 (en) | Method and apparatus for isolating a target bioentity from a biological sample | |
| US10704074B2 (en) | Method for detecting organisms in a diluted sample | |
| Ghosh Dastider et al. | Low concentration E. coli O157: H7 bacteria sensing using microfluidic MEMS biosensor | |
| Duarte et al. | Semi-quantitative method for streptococci magnetic detection in raw milk | |
| Cai et al. | A fluidic device for immunomagnetic separation of foodborne bacteria using self-assembled magnetic nanoparticle chains | |
| CN110093254A (en) | It is a kind of for fast Acquisition or detect cell micro-fluidic chip and method | |
| Jung et al. | Continuous, rapid concentration of foodborne bacteria (Staphylococcus aureus, Salmonella typhimurium, and Listeria monocytogenes) using magnetophoresis-based microfluidic device | |
| CN111617814A (en) | Microfluidic chip for detecting living organisms in water body and detection method | |
| Yang et al. | Separating and detecting escherichia coli in a microfluidic channel for urinary tract infection applications | |
| JP4957893B2 (en) | Isolation technology using integrated microwell array | |
| US11891594B2 (en) | Methods and apparatus for separating live from dead organisms in a sample | |
| Abdullah et al. | MEMS based impedance biosensor for rapid detection of low concentrations of foodborne pathogens | |
| Li et al. | Application of vertical electrodes in microfluidic channels for impedance analysis | |
| Wang et al. | Microfluidic sampling and biosensing systems for foodborne Escherichia coli and Salmonella | |
| CN212263293U (en) | Micro-fluidic chip for detecting living organisms in water body | |
| Tian et al. | Microfluidic diagnostic systems for the rapid detection and quantification of pathogens | |
| CN111569962A (en) | Protein enrichment method based on dielectrophoresis and multi-biological-probe rapid detection system | |
| CN111458392A (en) | Field-based biosensors for detecting full-cell bacteria and biosensors group including the same | |
| WO2000039577A1 (en) | Contaminant detection | |
| Sy et al. | Label-free and culture-free quantification of lactic acid bacteria using ring-shaped interdigitated electrodes | |
| Babaie | Magnetic microfluidic platform for bacteria isolation and detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |