CN108287236A - A kind of biological sensor and its application based on High-gradient Magnetic separation and quantum dot - Google Patents
A kind of biological sensor and its application based on High-gradient Magnetic separation and quantum dot Download PDFInfo
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- CN108287236A CN108287236A CN201711191273.9A CN201711191273A CN108287236A CN 108287236 A CN108287236 A CN 108287236A CN 201711191273 A CN201711191273 A CN 201711191273A CN 108287236 A CN108287236 A CN 108287236A
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Abstract
The present invention provides a kind of biological sensor and its application based on High-gradient Magnetic separation and quantum dot.The biological sensor of the present invention contains high-gradient magnetic separation unit and fluorescence detecting system, and high-gradient magnetic separation unit is made of annular magnet array, magnet holder, circular ring path and iron shot chain；Annular magnet array and magnet holder constitute toroidal magnetic field generator, the mutually exclusive assembling of each adjacent two magnet；The inner capillary tube and outer capillary that circular ring path is nested together by two form, two capillaries are nested in after coaxially fixing in the concentric circles of annular magnet array, the iron shot chain of soft magnetic materials is filled in inner capillary tube, the iron shot chain can be magnetized by toroidal magnetic field, nanometer magnetic bead is set to be evenly distributed in circular ring path, for capturing object.The biological sensor of the present invention can be used for quantitative detection microorganism, and easy to operate, separative efficiency is high, and sensitivity is good, and the quantitative detection of a small amount of target bacteria in food samples can be realized in 2 hours.
Description
Technical field
The present invention relates to technical field of biological, more particularly, to one kind based on High-gradient Magnetic separation and quantum dot
Microorganism detection method.
Background technology
Food-safety problem is by the extensive concern of international community in recent years, and in China, pathogenic microorganism is in causing food
The main factor of poison, has seriously threatened people's health, and cause huge economic loss.Prevent and controls food-borne
One key of illness outbreak is the rapid screening of microbial contamination food.Existing method of detecting bacterium mainly has cultivation
(for bacterium) or partition method (for virus), immunological method (such as ELISA), molecular biology method (such as PCR).It passes
The culture of system or partition method are goldstandard method, accuracy and high sensitivity, but time-consuming；ELISA detection times are shorter, specifically
Property it is preferable, easily realize high-throughput, but sensitivity is relatively low and false positive rate is higher；PCR detection times are also shorter, and sensitivity is higher,
But nucleic acid extraction is complicated for operation, and needs expensive instrument and equipment and technical professional.
Biological sensor is a kind of new bio detection technique, has high sensitivity, inspection easy to operate and non-contact
The advantages that survey, is used for the pathogenic microorganism examination by wide coverage.Quantum dot is a kind of common fluorescent material, has exciting light
The features such as spectrum width, emission spectrum are narrow, Stokes shift is larger, photochemical stability is high and fluorescence lifetime is long, is usually used in sensitive
Biological detection analysis.Due to many actual sample, such as food, excrement, there is very complicated background, easily cause glitch,
Therefore object is separated from complex sample solution is necessary, and a key of urgent need to resolve at present
Problem.Traditional filtration method and centrifugal process is that size based on object and quality are detached, without specificity, and in recent years
The immunomagnetic separation to grow up is to be based on antigen-antibody immune response, using immunomagnetic beads and externally-applied magnetic field by object from
It separates and is enriched in sample background, although with specificity, this method is typically only capable to processing corpusculum product sample, and base
All it is manual operation in sheet, it is more demanding to operating personnel.Therefore, it is higher, specific preferable big to develop a kind of separative efficiency
Volume samples microorganism separation method is simultaneously combined with Poul Dorset Sheep, the higher biological sensor of sensitivity applied to micro-
Biological detection is of great significance.
Invention content
(1) technical problems to be solved
The technical problem to be solved by the present invention is to study a kind of large volume sample object object specifically separation and enrichment method, and
It is combined with fluoroscopic examination, detection sensitivity and the automation mechanized operation for improving microorganism or other objects are horizontal, and shorten inspection
Survey the time.
(2) technical solution
To solve the above-mentioned problems, the high-gradient magnetic field of this method design can carry out substantially product sample and Magneto separate, behaviour is immunized
It is simpler to make step；It is micro- for quickly detecting the present invention provides a kind of biological sensor based on High-gradient Magnetic separation
Biology, the biological sensor contain high-gradient magnetic separation unit, the high-gradient magnetic field separator by annular magnet array,
Magnet holder, circular ring path and iron shot chain composition；
Wherein, annular magnet array and magnet holder constitute toroidal magnetic field generator, and the annular magnet array is by multiple
The identical annular magnet composition of specification, and the axis arranged adjacent centered on the center of circle, each adjacent two annular magnet are in mutual
Repulsion state；
The inner capillary tube and outer capillary that the circular ring path is nested together by two form, and are filled in inner capillary tube
It is nested in the concentric circles of annular magnet array after thering is soft magnetic materials iron shot chain arranged in a straight line, two capillaries coaxially to fix.
In embodiments herein, toroidal magnetic field generator includes 96 annular magnets and corresponds with fixed ring
The magnet holder of shape magnet is not limited to the application reality it will be appreciated by those skilled in the art that annular magnet number is the amount doesn't matter
Apply the quantity described in example.
The internal diameter of the annular magnet is slightly larger than the diameter of outer capillary, it is ensured that outer capillary can be embedded in annular magnet
The thickness of the close contact of diameter and holding and annular magnet, annular magnet is 0.5-1.3mm, preferably 1.0mm；The magnet holder
On be uniformly arranged the magnet slot for being useful for stationary annular magnet, be divided into 0.4-0.8mm, preferably 0.5mm between each two magnet slot.
In the embodiment of the present invention, the outer diameter of annular magnet is 5.5mm, and the thickness of internal diameter 2.8mm, annular magnet are
1.0mm；The long 130mm of magnet holder, the wide 9.5mm, each magnet slot thickness are 1.1mm, are divided between each two magnet slot
0.5mm。
The internal diameter of the inner capillary tube be 0.9-1.5mm, preferably 1.2mm, outer diameter 1.2-1.8mm, preferably 1.5mm, outside
The internal diameter of glass tube is 1.5-2.1mm, preferably 1.8mm, outer diameter 1.8-2.4mm, preferably 2.1mm；The inner capillary tube, outer hair
The material of tubule is quartz glass, simple glass or organic glass.
The diameter for the iron shot that the soft magnetic materials iron shot chain uses is less than the internal diameter of inner capillary tube, and the two gap is in 0.1-
Between 0.5mm, it is preferable that a diameter of 1.0mm of soft magnetic materials iron shot chain.
The soft magnetic materials iron shot chain can be magnetized by toroidal magnetic field, generate unique annular high-gradient magnetic field, avoid magnetic bead
Agglomerate is evenly dispersed in circular ring path.
The present invention also provides application of the above-mentioned biological sensor in microorganism detection.
The application, includes the following steps：
(1) magnetic bead of coupling protein G or albumin A being injected in circular ring path, the magnetic bead can be captured by high-gradient magnetic field,
It is evenly distributed in circular ring path, reinjects object bio-identification material, bio-identification material is incubated with Protein G or albumin A
In conjunction with rear, formation immunomagnetic beads, the Excess antibody of cleaning removing later；
(2) sample solution containing object is injected into circular ring path, the object bio-identification material on magnetic bead is caught
Object is obtained, cleaning later removes sample background, the magnetic bead for being purified and being enriched with-object complex；
(3) it is injected into the circular ring path and quantum dot is immunized, the magnetic bead-object complex is sent out with immune quantum dot
Raw immune combination, forms magnetic bead-object-quantum dot compound, and cleaning later removes extra immune quantum dot；
(4) circular ring path is taken out from the generator of toroidal magnetic field, and by magnetic bead-object-quantum dot compound from circle
It develops and recycles in ring channel, be detected later using fluorescence detecting system, obtain fluorescence intensity level, finally utilize mark
Determine the concentration that curve calculates object.
Wherein, the coupling protein G of step (1) or the magnetic bead of albumin A are dissolved in phosphate buffer, will with accurate peristaltic pump
In its high flow rate injection circular ring path with 1.1-1.3mL/min.Wriggling pump type used in the embodiment of the present application is T60-S2＆
WX10-14-A。
In step (1), Protein G or albumin A can be specifically bound with the Fc sections of object antibody, form immunomagnetic beads；Note
Enter ultrapure water and removes extra antibody.
Object bio-identification material described in step (1) is the monoclonal antibody of object, aptamer, bacteriophage
Or agglutinin.In embodiments herein, select the monoclonal antibody of object as object bio-identification material.
Step (2) injects the sample solution containing object into circular ring path, recycles 3 times.Using on immunomagnetic beads
The antibody captures object, forms magnetic bead-object complex；It injects ultra-pure water punching cleaning and removes other microorganisms
And impurity.
The step (3) includes the following steps：
1) preparation of the immune quantum dot is that first the quantum dot by streptavidin and biotinylated antibody carry out
Mixing is incubated, then is centrifuged off Excess antibody；
2) immune quantum dot is injected into circular ring path, after being incubated with magnetic bead-object complex, forms magnetic bead-object-
Quantum dot compound；It injects ultra-pure water punching cleaning and removes extra immune quantum dot.
The object identification material and the biotinylated antibody are different from the binding site of object, namely：Exempt from
The antibody modified on the antibody and the immune quantum dot modified on epidemic disease magnetic bead is different from the binding site of object, is two and matches
To antibody.
(3) advantageous effect
Realize the special separation of object in substantially product sample, have higher separative efficiency and detection sensitivity and
Better simply operating procedure can realize the quantitative detection of a small amount of microorganism in sample to be tested in 2 hours.
1, the present invention provides a kind of biological sensor based on high-gradient magnetic field separation, there is higher object
Separative efficiency, detection sensitivity is high, and method is simple to operation.
2, high-gradient magnetic separation unit of the invention can be such that magnetic bead is evenly distributed in circular passage, avoid immune magnetic
Pearl agglomerate, is greatly improved object separative efficiency, and the separative efficiency of object is higher than the separation of traditional magnetic separator
Efficiency, it is often more important that can be used for substantially product sample separation；
3, detection speed can be improved using the biological sensor of the present invention, large volume sample can be realized in 2 hours
The quantitative detection of a small amount of microorganism in this；
4, the object in milk authentic sample is detected using biological sensor of the present invention, the obtained rate of recovery is
95.92%~108.15%.
Description of the drawings
Fig. 1 is that the present invention is based on the biological sensors of High-gradient Magnetic separation and quantum dot quickly to detect microbial process
Flow chart.
Fig. 2 a are the circular ring path schematic diagram in the high-gradient magnetic separation unit of biological sensor of the present invention.
Fig. 2 b are the high-gradient magnetic field separator longitudinal section that biological sensor of the present invention removes magnet holder.
Fig. 2 c are the high-gradient magnetic separation unit cross-sectional view that biological sensor of the present invention removes magnet holder.
Fig. 2 d are for high-gradient magnetic separation unit in the present invention with COMSOL softwares to magnetic field simulation figure.
Fig. 2 e are that high-gradient magnetic separation unit sample passes through magnetic field gradient variation diagram in channel in the present invention.
Fig. 2 f are biological sensor high-gradient magnetic separation unit pictorial diagram of the present invention.
Fig. 3 is the operation principle signal of the biological sensor based on High-gradient Magnetic separation and quantum dot in the present invention
Figure.
Fig. 4 a are biosensor of the present invention to magnetic bead dosage optimization result figure.
Fig. 4 b are biosensor of the present invention to sample introduction passage flowing rate result figure.
Fig. 4 c are high-gradient magnetic separation unit in the present invention to various concentration (102-104CFU/mL) large intestine in sample solution
Bacillus O157：The capture result of H7.
Fig. 5 a are to capture the identical number in different volumes (1mL, 10mL) PBS respectively using biological sensor of the present invention
Amount (104CFU Escherichia coli O 157)：The testing result of H7.
Fig. 5 b are that biological sensor of the present invention is used for same concentrations (104CFU/mL Escherichia coli O 157)：H7 (makees
For target bacteria) and salmonella typhimurium and Listeria monocytogenes (as interference bacterium) testing result.
Fig. 6 a are that biological sensor of the present invention is used for various concentration (8.9 × 100-8.9×105CFU/mL large intestine)
Bacillus O157：The spectral detection result of H7.
Fig. 6 b are the calibration curve of biological sensor of the present invention.
Fig. 6 c are that biological sensor of the present invention is used for various concentration (1.1 × 101-1.1×105CFU/mL large intestine)
Bacillus O157：H7 manually adds the testing result of milk sample.
Specific implementation mode
With reference to the accompanying drawings and examples, the specific implementation mode of the present invention is described in further detail.Implement below
Example is not limited to the scope of the present invention for illustrating the present invention.Biochemical reagents, the soft magnetic materials iron shot chain of the present invention, interior/
Outer capillary, annular magnet are commercially available.
Protein G (a kind of cell wall protein that G type streptococcus is isolated) of the present invention or albumin A are (golden yellow from A types
A kind of cell wall protein that color staphylococcus is isolated) it is known in the art shared biomaterial, it is commercially available.This hair
The Protein G and albumin A of bright embodiment are purchased from Shanghai Yaxin Biotech Co., Ltd..
The structure of biological sensor of the embodiment 1 based on High-gradient Magnetic separation and quantum dot
The biological sensor based on High-gradient Magnetic separation and quantum dot of the present invention, including High-gradient Magnetic separation dress
It sets, fluorescence detecting system, peristaltic pump, water conservancy diversion connecting tube etc..
The high-gradient magnetic separation unit is made of magnet holder, annular magnet array, circular ring path and iron shot chain；
Wherein, annular magnet array and magnet holder constitute toroidal magnetic field generator, and all annular magnets are identical, and
The axis arranged adjacent centered on the center of circle, each adjacent two magnet is in mutually exclusive state；
The inner capillary tube and outer capillary that circular ring path is nested together by two form, filled with straight in inner capillary tube
The soft magnetic materials iron shot chain of line arrangement, two capillaries are nested in after coaxially fixing in the concentric circles of annular magnet array.
The toroidal magnetic field generator includes 96 (the amount doesn't matter, to be not limited to quantity) annular magnets and magnet holder.
The outer diameter of the annular magnet is 5.5mm, and the thickness of internal diameter 2.8mm, annular magnet are 1.0mm；The magnet
Holder long 130mm, wide 9.5mm, each magnet slot thickness are 1.1mm, and 0.5mm is divided between each two magnet slot.
Wherein, the internal diameter of inner capillary tube is 1.2mm, and the internal diameter of outer diameter 1.5mm, outer capillary are 1.8mm, and outer diameter is
2.1mm。
A diameter of 1.0mm of the soft magnetic materials iron shot chain.
The soft magnetic materials iron shot chain can be magnetized by toroidal magnetic field, generate unique annular high-gradient magnetic field, avoid magnetic bead
Agglomerate is evenly dispersed in circular ring path.
The fluorescence detecting system is made of spectrometer, light source, optical fiber and detection cell.Spectrometer is public using Ocean Optics
The USB4000-UV-VIS spectrometers of department, light source use USB-LS-450 blue light sources, optical fiber to use QR400-7-UV, detection cell
It is self-control suprasil small container.
Fig. 1 quickly detects microbial process for a kind of biological sensor based on High-gradient Magnetic separation in the present invention
Flow chart, Fig. 2 are the high-gradient magnetic separation unit provided in the present invention, logical in annulus by COMSOL softwares to magnetic field simulation
The mean intensity of each of road annular high-gradient magnetic field is 0.46T, and average gradient 590T/m can efficiently capture magnetic
Pearl, to improve object capture rate.Fig. 3 is a kind of fluorescence life based on high-gradient magnetic field separation and quantum dot in the present invention
The operation principle schematic diagram of object sensor, method include the following steps：
(1) magnetic bead of coupling protein G is injected in the circular ring path, the magnetic bead can be caught by the high-gradient magnetic field
It obtains, is evenly distributed in circular ring path, the antibody (or other bio-identification materials) for reinjecting anti-object is incubated with Protein G
After educating combination, immunomagnetic beads are formed, cleaning later removes Excess antibody；This step keeps immunomagnetic beads uniform by high-gradient magnetic field
Dispersion, increases the area contacted with object, to improve capture rate；
(2) sample solution containing object is injected into the circular ring path, the antibody on the magnetic bead will be caught
Object is obtained, cleaning later removes sample background, the magnetic bead for being purified and being enriched with-object complex；This step is narrow
Circular ring path improves the collision probability of object and antibody, improves immunocapture efficiency, realizes the enrichment of object, from
And improve the sensitivity of detection；
(3) it is injected into the circular ring path and quantum dot is immunized, the magnetic bead-object complex will be with immune quantum dot
Immune combination occurs, forms magnetic bead-object-quantum dot compound, cleaning later removes extra immune quantum dot；
(4) circular ring path is taken out from the toroidal magnetic field generator, and by the magnetic bead-object-quantum
Point compound is developed and is recycled from the circular ring path, is detected, is obtained using the fluorescence detecting system later
Fluorescence intensity level finally utilizes calibration curve to calculate the concentration of object.
The step (1) specifically includes following steps：
Protein G and carboxyl magnetic bead are coupled using EDC methods, prepare the magnetic bead of the Protein G modification, and will be described
The magnetic bead of Protein G modification is dissolved in phosphate buffer (PBS), logical with the flow velocity injection annulus of 1.2mL/min using peristaltic pump
In road；Then object antibody is injected into circular ring path, utilizes the Fc of Protein G and the immunoglobulin of antibody section specificity knots
It closes, forms immunomagnetic beads；
The immunomagnetic beads formed in this step are mediated by Protein G, and magnetic bead and antibody coupling are made, and are formed and magnetic is immunized
Pearl.In addition, due to the presence of high-gradient magnetic field, the Fc sections of antibody are connected with magnetic bead, the Fab section of antibody can towards channel center,
The probability that object is combined with antibody is increased, and then improves immune response efficiency；
It injects ultra-pure water cleaning and removes extra antibody.
Further, the step (2) specifically includes following steps：The sample containing object is injected into circular ring path
Solution recycles 3 times.Object is captured using the antibody on immunomagnetic beads, it is compound to form the magnetic bead-object
Body；It injects ultra-pure water punching cleaning and removes other microorganisms and impurity.
The step (3) specifically includes following steps：1) preparation of the immune quantum dot is first by streptavidin
Quantum dot and biotinylated antibody carry out mixing incubation, then be centrifuged off Excess antibody；
2) immune quantum dot is injected into circular ring path, after being incubated with magnetic bead-object complex, forms magnetic bead-object-
Quantum dot compound；It injects ultra-pure water cleaning and removes extra immune quantum dot.
The step (4) specifically includes following steps：
Toroidal magnetic field generator is removed, with phosphate buffer by magnetic bead-object-quantum dot complex from circular ring path
In go out；Then magnetic bead-object-quantum dot complex solution is detected with the fluorescence detecting system, obtains fluorescence
Intensity value；According to the calibration curve of fluorescence intensity level and target concentration, the concentration of object in sample solution is calculated.
Further, the antibody modified on the antibody and the immune quantum dot modified on the immunomagnetic beads is pairing
, it is different from the binding site of object, therefore the combination of immunomagnetic beads and immune quantum dot and object is not influenced.It is above-mentioned anti-
Body can also be replaced with other biological identification elements such as aptamer, bacteriophage, agglutinin.
Embodiment 2 quantitatively detects Escherichia coli O 157 using the biological sensor of the present invention：H7
The present embodiment is with Escherichia coli O 157：H7 is as object model, Escherichia coli O 157：H7 is common food-borne
Pathogen is found in beef, chicken, milk, vegetables and fruit etc., it might even be possible to temperature down to 4 DEG C, pH down to
It survives under conditions of 2.5.It has very strong toxicity, its infective dose is down to 10CFU/mL.Escherichia coli O 157：H7 infects
Person may suffer from watery diarrhea and hemolytic uremic syndrome etc., or even dead.Therefore, in order to prevent and control Escherichia coli
O157：The food origin disease that H7 causes, there is an urgent need to establish a kind of quick, sensitive Escherichia coli O 157：H7 detection methods, under
Face embodiment provides solution to the problem.
Material used in the present embodiment mainly has：
Phosphate buffer (PBS, 10mM, pH 7.4)；180nm carboxyl magnetic beads；1- (3- dimethylamino-propyls) -3- ethyls
Carbodiimide hydrochloride (EDCHCl)；Hydroxy thiosuccinimide (NHSS)；Luria-Bertani culture mediums (LB)；Ox
Haemocyanin (BSA)；The quantum dot (QDs) of streptavidin；Protein G (PG) etc..
It is prepared by PG- magnetic beads：
(a) activated magnetic beads：Add 9mL PBS (0.01M, pH 7.4) after glass centrifuge tube, adds 1mL magnetic beads (10mg/
ML), wash 3 times, be then resuspended in 10mL PBS.2.88mg EDC and 3.26mg NHSS are added, is uniformly mixed, is placed in mixed
(15rpm) is rotated on even instrument, room temperature activates 1h.
(b) magnetic bead and PG couplings：After the above-mentioned magnetic bead of 10mL PBS washings 3 times, magnetic bead is resuspended in 10mL PBS,
0.8mg PG (being dissolved in PBS, 5mg/mL) are added, are placed on shaking table, are kept for 37 DEG C, are coupled 2h.
(c) it closes：A concentration of 1% BSA solution is added, room temperature closes 45min (15rpm).Magnetic captures magnetic bead, utilizes
10mL PBS are washed 3 times, are recycled 5mL PBS that PG- magnetic beads are resuspended, are obtained the PG- magnetic beads of a concentration of 2mg/ml, be placed in 4 DEG C of guarantors
It deposits.
The preparation of immune quantum dot：
20 μ L biotinylated more anti-(0.4-0.5mg/ml is purchased from Biodesign companies, Saco, ME, US) and 20 μ L
QDs (1 μM, be purchased from Ocean Nanotech companies, San Diego, CA, US) mixing, is added 160 μ L PBS, incubation at room temperature
30min (15rpm), obtains immune quantum dot, and centrifugation (speed 70000rpm) removes extra antibody, is placed in 4 DEG C of preservations.
Illustrate that biological sensor is to Escherichia coli O 157 made from the embodiment of the present invention 1 below：The specific detection of H7
Steps are as follows：
(a) circular ring path is cleaned：Circular ring path is placed in the generator of toroidal magnetic field, is then connect with peristaltic pump, successively
Injection ultra-pure water, 75% alcohol and PBS are cleaned.
(b) immunomagnetic beads are formed：35 μ L PG magnetic beads (180nm, 2mg/ml) are taken, 165 μ L PBS are added, utilize peristaltic pump
It is injected in circular ring path, is recycled 3 times, magnetic bead is uniformly captured in circular ring path with 1.2mL/min flow velocitys；Then with
The flow velocity injection 5mL PBS of 0.36mL/min clean magnetic bead background, then by 30 μ L Escherichia coli O 157s：Antibody (the 0.73mg/ of H7
ML it) is injected in circular ring path with 0.36mL/min flow velocitys, stationary incubation 30min, forms immunomagnetic beads, and clear using 5mL PBS
Extra antibody is removed in washout.
(c) target bacteria is captured：With the flow velocity injection 1mL various concentrations (10 of 0.36mL/min1-105CFU/mL large intestine)
Bacillus O157：H7 is recycled 3 times, and target bacteria will be trapped in circular ring path, and is removed sample using 5mL PBS cleanings and carried on the back
Scape.
Fig. 4 a and Fig. 4 b be the present invention to magnetic bead dosage, sample introduction passage flow parameters optimization as a result, after optimization
It selects under optimum condition, 4c is high-gradient magnetic separation unit of the present invention to various concentration (102-104CFU/mL) big in sample solution
Enterobacteria O157：The capture result of H7, it can be seen that：It being capable of efficient capture large intestine using the high-gradient magnetic separation unit of the present invention
Bacillus O157：H7.
(d) incorporating quantum point：200 μ L, which are injected, with the flow velocity of 0.12mL/min is immunized quantum dot, stationary incubation 30min, and
Extra immune quantum dot is removed using 5mL PBS cleanings.
(e) fluoroscopic examination：Toroidal magnetic field generator is removed, 100 μ L PBS (10mM) are injected with the flow velocity of 0.60mL/min
Magnetic bead-bacterium-quantum dot complex is gone out into circular ring path, and is recovered in progress fluorescence intensity detection in quartzy small container, is led to
Cross fluorescence intensity level and Escherichia coli O 157：The calibration curve of H7 concentration calculates Escherichia coli O 157：The concentration of H7.
Fig. 5 a are that the method for the present invention is captured using biological sensor of the present invention respectively at different volumes (1mL, 10mL)
Identical quantity (10 in PBS4CFU Escherichia coli O 157)：The testing result of H7；Fig. 5 b are that the method for the present invention is used for same concentrations
(104CFU/mL Escherichia coli O 157)：H7 (as target bacteria) and salmonella typhimurium and Listeria monocytogenes (as
Interfere bacterium) testing result, it can be seen that：Using the biological sensor of the present invention, Escherichia coli can be effectively captured
O157：H7, but only have faint non-specific binding to salmonella typhimurium and Listeria monocytogenes.
Fig. 6 a biological sensors of the present invention are used for various concentration (8.9-8.9 × 105CFU/mL Escherichia coli)
O157：The spectral detection result of H7；Fig. 6 b are the calibration curve of biological sensor of the present invention；Fig. 6 c give birth to for fluorescence of the present invention
Object sensor is used for various concentration (1.1 × 101-1.1×105CFU/mL Escherichia coli O157) manually adds milk sample
Testing result, it can be seen that：In circular ring path, immunomagnetic beads, Escherichia coli O157 and immune quantum dot can combine
For magnetic bead-bacterium-quantum dot complex, work as Escherichia coli O 157：When H7 concentration increases, fluorescence intensity level also accordingly increases, and
Preferable linear relationship is presented.
Embodiment of above is merely to illustrate the present invention rather than limitation of the present invention.Although with reference to embodiment to this hair
It is bright to be described in detail, it will be understood by those of ordinary skill in the art that, to technical scheme of the present invention carry out it is various combination,
Modification or equivalent replacement, without departure from the spirit and scope of technical solution of the present invention, the right that should all cover in the present invention is wanted
It asks in range.
Claims (10)
1. a kind of biological sensor based on High-gradient Magnetic separation and quantum dot, which is characterized in that contain High-gradient Magnetic point
From device, which is made of annular magnet array, magnet holder, circular ring path and iron shot chain；
Wherein, annular magnet array and magnet holder constitute toroidal magnetic field generator, and the annular magnet array is by multiple specifications
Identical annular magnet composition, and the axis arranged adjacent centered on the center of circle, each adjacent two annular magnet are in mutually exclusive
State；
The inner capillary tube and outer capillary that the circular ring path is nested together by two form, filled with straight in inner capillary tube
The soft magnetic materials iron shot chain of line arrangement, two capillaries are nested in after coaxially fixing in the concentric circles of annular magnet array.
2. biological sensor as described in claim 1, which is characterized in that the internal diameter of the annular magnet is slightly larger than outer hair
The diameter of tubule, it is ensured that outer capillary can be embedded in annular magnet internal diameter and keep the close contact with annular magnet, annular magnetic
The thickness of iron is 0.5-1.3mm, preferably 1.0mm；The magnet for being useful for stationary annular magnet is uniformly arranged in the magnet holder
Slot is divided into 0.4-0.8mm, preferably 0.5mm between each two magnet slot.
3. biological sensor as described in claim 1, which is characterized in that the internal diameter of the inner capillary tube is 0.9-
The internal diameter of 1.5mm, preferably 1.2mm, outer diameter 1.2-1.8mm, preferably 1.5mm, outer glass pipe are 1.5-2.1mm, preferably
1.8mm, outer diameter 1.8-2.4mm, preferably 2.1mm；The inner capillary tube, outer capillary material be quartz glass, common glass
Glass or organic glass.
4. biological sensor as described in any one of claims 1-3, which is characterized in that the soft magnetic materials iron shot chain uses
Iron shot diameter be less than inner capillary tube internal diameter, the two gap is between 0.1-0.5mm.
5. any biological sensors of claim 1-4, which is characterized in that when the circular ring path is placed in the ring
When in shape magnetic field generating, the iron shot chain will be magnetized, and high-gradient magnetic field is generated around the iron shot chain, and application is such as
Under：
(1) magnetic bead of coupling protein G or albumin A being injected in the circular ring path, the magnetic bead can be captured by high-gradient magnetic field,
It is evenly distributed in circular ring path, reinjects object bio-identification material, bio-identification material is incubated with Protein G or albumin A
In conjunction with rear, formation immunomagnetic beads, the Excess antibody of cleaning removing later；
(2) sample solution containing object is injected into the circular ring path, the object bio-identification material on magnetic bead is caught
Object is obtained, cleaning later removes sample background, the magnetic bead for being purified and being enriched with-object complex；Object identifies material
Material can be the monoclonal antibody of object；
(3) it is injected into the circular ring path and quantum dot is immunized, the magnetic bead-object complex is exempted from immune quantum dot
Epidemic disease combines, and forms magnetic bead-object-quantum dot compound, and cleaning later removes extra immune quantum dot；
(4) circular ring path is taken out from the generator of toroidal magnetic field, and magnetic bead-object-quantum dot compound is led to from annulus
It develops and recycles in road, be detected later using fluorescence detecting system, obtain fluorescence intensity level, it is finally bent using calibration
The concentration of line computation object.
6. application of any biological sensors of claim 1-5 in detecting object.
7. application as claimed in claim 6, which is characterized in that include the following steps：
(1) magnetic bead of coupling protein G or albumin A is injected in circular ring path, the magnetic bead can be captured by high-gradient magnetic field, uniformly
It is distributed in circular ring path, reinjects object bio-identification material, bio-identification material and Protein G or albumin A hatching combination
Afterwards, immunomagnetic beads are formed, cleaning later removes Excess antibody；
(2) sample solution containing object, the object bio-identification material capture mesh on magnetic bead are injected into circular ring path
Object is marked, cleaning later removes sample background, the magnetic bead for being purified and being enriched with-object complex；
(3) it is injected into the circular ring path and quantum dot is immunized, the magnetic bead-object complex is exempted from immune quantum dot
Epidemic disease combines, and forms magnetic bead-object-quantum dot compound, and cleaning later removes extra immune quantum dot；
(4) circular ring path is taken out from the generator of toroidal magnetic field, and magnetic bead-object-quantum dot compound is led to from annulus
It develops and recycles in road, be detected later using fluorescence detecting system, obtain fluorescence intensity level, it is finally bent using calibration
The concentration of line computation object.
8. the use as claimed in claim 7, which is characterized in that the coupling protein G of step (1) or the magnetic bead of albumin A are dissolved in phosphorus
It, will be in its high flow rate injection circular ring path with 1.1-1.3mL/min with accurate peristaltic pump in phthalate buffer.
9. application as claimed in claim 7 or 8, which is characterized in that step (2) is injected into circular ring path contains object
Sample solution recycles 3-4 times, preferably 3 times.
10. the application as described in claim 7-9 is any, which is characterized in that step (3) the immune quantum dot is by strepto- parent
It is incubated and is formed with the quantum dot of elementization and the mixing of biotinylated antibody；The object bio-identification material and the biotin
The antibody of change is different from the binding site of object.
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