CN107677817A - A kind of salmonella typhimurium quick determination method based on immune magnetic Nano material photo-thermal effect - Google Patents
A kind of salmonella typhimurium quick determination method based on immune magnetic Nano material photo-thermal effect Download PDFInfo
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- CN107677817A CN107677817A CN201710756436.7A CN201710756436A CN107677817A CN 107677817 A CN107677817 A CN 107677817A CN 201710756436 A CN201710756436 A CN 201710756436A CN 107677817 A CN107677817 A CN 107677817A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention establishes a kind of salmonella typhimurium quick determination method based on immune magnetic Nano material photo-thermal effect, the standard curve between temperature increase and salmonella typhimurium number is established using the photo-thermal effect of immune magnetic Nano material, temperature increase caused by sample can be brought into standard curve and obtain the number of bacteria in sample.Simultaneously, utilize the magnetic and photo-thermal effect of immune magnetic Nano material, realize effectively integrating for concentration and separation detection sample three links of inactivation, this method is sensitive, safe, quick, portable, cost is low, experiments verify that, salmonella typhimurium method lowest detection based on immune magnetic Nano material photo-thermal effect is limited to 300 bacteriums, and temperature increase Δ T and number of bacteria linear dependency relation in the range of 300~1000, therefore the present invention has good actual application prospect, can be applied to the actual quick detection to salmonella typhimurium.
Description
Technical field
It is more particularly to a kind of based on immune magnetic Nano material photo-thermal effect the present invention relates to technical field of immunoassay
Salmonella typhimurium quick determination method.
Background technology
It is always that the World Health Organization and national governments pay much attention to by the microbial food-safety problem of food-borne pathogenic
Global problem, and food safety management and core and one of difficult point of control.Salmonellosis is that important people and animals are total to
One of illness, its cause of disease Salmonella enteric bacteria section, can cause food poisoning.
The detection method of current salmonella is typically both needed to amplification step, with increase cause of disease can recall rate, waste time and energy,
Need to complete for 4~7 days, can not in time, the content of Fast Evaluation Salmonella in Food.Resist currently with thalline or flagellum
The immunological method of former specific antibody is the main direction of studying of quick determination method.The salmonella having built up is immunized
Learning detection method has ELISA, fluorescent immune method, radioimmunology, immunosensor etc., and such method can be with enzyme mark
Plate, test strips, film or electrode etc. are used as immobilization carrier.Wherein, the method based on test strips or film is the most simple and quick, without
Large-scale instrument, suitable for qualitative or semi-quantitative analysis.Because most samples to be tested are feminine gender, for the quick fixed of large sample size
Property or sxemiquantitative examination can greatly improve detection efficiency, and therefore, it is quick that such quick determination method is highly suitable for large sample size
Examination.But because such method is mostly using visual or simple detection device, sensitivity is still relatively low, is only improved spirit
Sensitivity, then with salmonella in the food samples that complex matrices are just can be suitably used for for being enriched with the pre-treatment step of purification
Rapid screening.
As the above analysis, most pathogenic bacteria detection methods are by concentration and separation-detection-experiment inactivation point at present
Being carried out for three links, step is more, and time-consuming, and most reports on analysis time pertain only to detect this link at present,
And 2h is no less than the time required to whole analysis process is inactivated from sample treatment to sample, if need to increase collarium section will be no less than 10 h,
And the detection sensitivity of existing pathogenic bacteria detection method still has much room for improvement.In view of this, it is a kind of to salmonella spirit to need offer badly
Quick, safe, quick, portable, inexpensive detection method.
The content of the invention
For above-mentioned the deficiencies in the prior art, inventor provides a kind of mouse based on immune magnetic Nano material photo-thermal effect
Salmonella typhi quick determination method, this method are realized to salmonella typhimurium simultaneously using immune magnetic Nano material
Capture, detection and inactivation, while using temperature change caused by laser irradiation immune magnetic Nano material, establish temperature rise
The standard curve of value and salmonella typhimurium number, realizes the quantitative detection to salmonella typhimurium.
Specifically, the present invention relates to following technical scheme：
The first aspect of the invention, immune magnetic Nano material is disclosed in salmonella typhimurium quick detection
Using.The immune magnetic Nano material is added in salmonella typhimurium, specifically, by carboxyl magnetic bead subscript
Anti- salmonella typhimurium antibody is remembered so as to which immune magnetic Nano material be made, and then is coupled with salmonella typhimurium,
Realize and salmonella typhimurium is captured.
Wherein, the preparation method of the immune magnetic Nano material is：
1) carboxyl magnetic bead is taken, ultrasound, Magneto separate, is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature bar
Activated under part, then carry out Magneto separate, magnetic bead after must being activated after being cleaned repeatedly with PBS solution；
2) anti-salmonella typhimurium antibody is added into the magnetic bead after activation, reacts a period of time, magnetic under room temperature condition
Separation, is then cleaned, obtains immune magnetic Nano material repeatedly with PBS solution.
Wherein, the particle diameter of the carboxyl magnetic bead is 10~300nm (being preferably 200~300nm)；Obtained under the particle diameter
Immune magnetic Nano material photo-thermal effect is optimal, while the Magneto separate time is short, and low to the non-specific adsorption of bacterium.
The second aspect of the invention, disclose a kind of mouse typhus sramana based on immune magnetic Nano material photo-thermal effect
Salmonella quick determination method, its specific steps include：
S1. carboxyl magnetic bead and anti-salmonella typhimurium antibody are coupled to obtain immune magnetic Nano material；
S2. immune magnetic Nano material step S1. being prepared is added in salmonella typhimurium, is incubated, film
Filtering, and be further purified with magnetic separation technique, obtain salmonella typhimurium-immune magnetic Nano material composite；
S3. salmonella typhimurium-immune magnetic Nano the material composite being trapped on film is irradiated with laser, produced
Temperature change, standard curve is established according to temperature increase and number of bacteria；Photo-thermal based on immune magnetic Nano material simultaneously
Effect, it is synchronous that salmonella typhimurium is inactivated.
Wherein, the specific preparation method of step S1. immune magnetic Nanos material is：
S1.1 takes carboxyl magnetic bead, ultrasound, Magneto separate, is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature
Under the conditions of activate, then carry out Magneto separate, magnetic bead after must being activated after being cleaned repeatedly with PBS solution；
S1.2 adds anti-salmonella typhimurium antibody into the magnetic bead after activation, reacts a period of time under room temperature condition,
Magneto separate, then cleaned repeatedly with PBS solution, obtain immune magnetic Nano material.
Wherein, the particle diameter of carboxyl magnetic bead described in step S1.1 is 10~300nm (being preferably 200~300nm)；The grain
The immune magnetic Nano material photo-thermal effect obtained under footpath is optimal, while the Magneto separate time is short, and to the non-specific adsorption of bacterium
It is low；
In step S2. the addition of immune magnetic Nano material be 9~10 μ g/1000 salmonella typhimuriums (preferably
For 10 μ g/1000 salmonella typhimuriums), immune magnetic Nano material addition is too low or too high, can cause photo-thermal
Effect is relatively low, influences experimental result；
Laser power is 2.5~2.8Wcm in the step S3.-2(preferably 2.5Wcm-2), wavelength 808nm, shine
The time is penetrated as 2~5 minutes (being preferably 2 minutes)；Irradiation power is too low, then immune magnetic Nano material warms are slow, extends inspection
Survey time and inactivation time；Irradiation power is too high, then immune magnetic Nano material warms are too high to produce destruction to filter membrane；
It is as follows that the specifically used formula of standard curve established according to temperature increase and number of bacteria in the step S3.：
Δ T=Δ T1- Δs T0
Wherein, Δ T is temperature increase
Δ T0 is the temperature increase after blank (being not added with salmonella typhimurium) sample laser pre-irradiation.
Δ T1 is the temperature increase after laser pre-irradiation after addition sample.
In order to eliminate the influence for being trapped in unnecessary immune magnetic Nano material on filter membrane, therefore blank (should be not added with mouse wound
Cold salmonella) temperature increase (Δ T0) after sample laser pre-irradiation is from the temperature after laser pre-irradiation after addition sample
Deducted in lift-off value (Δ T1)；
The invention also discloses application of the above-mentioned detection method in quick detection salmonella typhimurium, specifically, institute
Application mode is stated to be used for quickly detecting to salmonella typhimurium in drinking water.
Beneficial effects of the present invention：
The present invention establishes a kind of salmonella typhimurium quick detection based on immune magnetic Nano material photo-thermal effect
Method, established using the photo-thermal effect of immune magnetic Nano material between temperature increase and salmonella typhimurium number
Standard curve, temperature increase caused by sample can be brought into standard curve and obtain the number of bacteria in sample.Meanwhile utilize
The magnetic and photo-thermal effect of immune magnetic Nano material, realize the effective of concentration and separation-detection-sample three links of inactivation
Integrate, i.e., realize that the photo-thermal of the magnetic enrichments of pathogenic bacteria, Opto-thertnal detection and sample inactivates three kinds of work(simultaneously using a kind of nano material
Can, the bulk analysis time is foreshortened within 1.5h；
This method is sensitive, safe, quick, portable, cost is low, experiments verify that, based on immune magnetic Nano material photo-thermal
The salmonella typhimurium method lowest detection of effect is limited to 300 bacteriums, and temperature increase Δ T and number of bacteria are 300
Linear dependency relation in the range of~1000, therefore the present invention has good actual application prospect, can be applied to mouse typhus
The actual quick detection of salmonella.
Brief description of the drawings
Figure 1A is immune magnetic Nano material identification salmonella typhimurium schematic diagram；Figure 1B is based on immune magnetic Nano
The salmonella typhimurium capture of material photo-thermal effect and photo-thermal effect detection process schematic diagram；
Fig. 2 is photo-thermal effect figure of the different-grain diameter carboxyl magnetic bead under various concentrations；
Fig. 3 is the comparison figure of different-grain diameter carboxyl magnetic bead Magneto separate time；
Fig. 4 is different-grain diameter carboxyl magnetic bead to salmonella typhimurium non-specific adsorption figure；
Fig. 5 is that salmonella typhimurium is coupled immune magnetic Nano material transmission electron microscope picture；
Fig. 6 is that fluorescent staining salmonella typhimurium is coupled fluorescence immunoassay magnetic Nano material fluorogram；
Fig. 7 (A) is that different laser irradiation powers and carboxyl magnetic bead heat up graph of a relation, the different laser irradiation times of Fig. 7 (B) with
Carboxyl magnetic bead heating graph of a relation；
Fig. 8 is immune magnetic Nano material addition and its photo-thermal effect graph of a relation；
Fig. 9 (A) is PBS and Δ T in drinking water and the canonical plotting of salmonella number, and Fig. 9 (B) is in drinking water
The testing result figure of salmonella；The method specificity that Fig. 9 (C) is the present invention investigates figure, respectively for salmonella, large intestine
Bacillus and staphylococcus aureus are investigated.
Figure 10 is to be incubated carboxyl magnetic bead after laser difference exposure time to the bactericidal effect figure of salmonella typhimurium.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the application.It is unless another
Indicate, all technologies used herein and scientific terminology have to be led to the application person of an ordinary skill in the technical field
The identical meanings understood.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise odd number shape
Formula is also intended to include plural form, additionally, it should be understood that, when in this manual use term "comprising" and/or
During " comprising ", it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
As described in background technology, most pathogenic bacteria detection methods are that concentration and separation-detection-experiment is gone out
Living to be divided into three links progress, step is more, and time-consuming.
In view of this, in a kind of embodiment of the invention, there is provided one kind is based on immune magnetic Nano material photo-thermal
The salmonella typhimurium quick determination method of effect, its specific steps include：
S1. carboxyl magnetic bead and anti-salmonella typhimurium antibody are coupled to obtain immune magnetic Nano material；
S2. immune magnetic Nano material step S1. being prepared is added in salmonella typhimurium, is incubated, film
Filtering, and be further purified with magnetic separation technique, obtain salmonella typhimurium-immune magnetic Nano material composite；
S3. salmonella typhimurium-immune magnetic Nano the material composite being trapped on film is irradiated with laser, produced
Temperature change, standard curve is established according to temperature increase and number of bacteria；Photo-thermal based on immune magnetic Nano material simultaneously
Effect, it is synchronous that salmonella typhimurium is inactivated.
Wherein, the specific preparation method of step S1. immune magnetic Nanos material is：
S1.1 takes carboxyl magnetic bead, ultrasound, Magneto separate, is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature
Under the conditions of activate, then carry out Magneto separate, magnetic bead after must being activated after being cleaned repeatedly with PBS solution；
S1.2 adds anti-salmonella typhimurium antibody into the magnetic bead after activation, reacts a period of time under room temperature condition,
Magneto separate, then cleaned repeatedly with PBS solution, obtain immune magnetic Nano material.
Wherein, the particle diameter of carboxyl magnetic bead described in step S1.1 is 10~300nm (being preferably 200~300nm)；The grain
The immune magnetic Nano material photo-thermal effect obtained under footpath is optimal, while the Magneto separate time is short, and to the non-specific adsorption of bacterium
It is low；
In step S2. the addition of immune magnetic Nano material be 9~10 μ g/1000 salmonella typhimuriums (preferably
For 10 μ g/1000 salmonella typhimuriums), immune magnetic Nano material addition is too low or too high, can cause photo-thermal
Effect is relatively low, influences experimental result；
Laser power is 2.5~2.8Wcm in the step S3.-2(preferably 2.5Wcm-2), wavelength 808nm, shine
The time is penetrated as 2~5 minutes (being preferably 2 minutes)；Irradiation power is too low, then immune magnetic Nano material warms are slow, extends inspection
Survey time and inactivation time；Irradiation power is too high, then immune magnetic Nano material warms are too high to produce destruction to filter membrane；
It is as follows that the specifically used formula of standard curve established according to temperature increase and number of bacteria in the step S3.：
Δ T=Δ T1- Δs T0
Wherein, Δ T is temperature increase
Δ T0 is the temperature increase after blank (being not added with salmonella typhimurium) sample laser pre-irradiation.
Δ T1 is the temperature increase after laser pre-irradiation after addition sample.
In order to eliminate the influence for being trapped in unnecessary immune magnetic Nano material on filter membrane, therefore blank (should be not added with mouse wound
Cold salmonella) temperature increase (Δ T0) after sample laser pre-irradiation is from the temperature after laser pre-irradiation after addition sample
Deducted in lift-off value (Δ T1).
Mode by the following examples is further elaborated to the present invention.
Embodiment
1. test material and equipment
1.1 main materials and reagent
The reagent of table 1 and medicine
1.2 key instruments and equipment
Table 3-2 instruments and equipment
Table 3-2Equipments and Instruments
2 related solutions are prepared
EDC/NHS solution is prepared：EDC 9.6mg is weighed respectively, NHS 9.6mg is dissolved in 3mL secondary waters.
0.01mol·L-1Phosphate buffer solution is prepared：Na is weighed respectively2HPO4·12H2O 13.76g、NaH2PO4·2
H2O 1.79g, NaCl 9.00g, add deionized water to be settled to 1000mL.
SYBR Green Ι standard solutions are prepared：SYBR Green Ι stostes are diluted 100 times with DMSO, -20 DEG C of guarantors
Deposit.
EDTA-2Na is prepared using liquid：EDTA-2Na 18.61g are weighed, add sterile purified water to be settled to 100mL.
LB fluid nutrient mediums are prepared：Tryptone 10g, yeast extract 5g, NaCl 10g are weighed respectively, use deionized water
1000mL is settled to, adjusts pH to 7.0.
Beef-protein medium is prepared：Beef extract 3g, peptone 10g, NaCl 5g are weighed respectively, use deionized water
1000mL is settled to, adds 20g agar.
Culture, preparation and the fluorescent staining method of 3 salmonella typhimuriums
The culture of 3.1 salmonella typhimuriums
With oese, picking salmonella typhimurium single bacterium colony is placed in LB fluid nutrient mediums from inclined-plane, 37 DEG C of vibration trainings
Bacterium solution is collected after supporting 8h.
The preparation of 3.2 salmonella typhimuriums
The bacterium solution 1mL of collection is taken to carry out gradient dilution (10,102、103、104、105、106), each dilution factor bacterium solution is taken respectively
25 μ L even spreads are counted after cultivating 16h in 37 DEG C of incubators to beef-protein medium surface.
Each dilution factor bacterium solution 1mL, 4 DEG C of 3000rmin are taken respectively-1Centrifuge 5min, abandon supernatant (remove culture medium and other
Impurity), PBS is centrifuged again after redissolving.Above-mentioned centrifugally operated is repeated, abandons supernatant, it is stand-by that 10 μ L PBS redissolve precipitation.
The fluorescent staining method of 3.3 salmonella typhimuriums
1mL bacterium solutions are taken in polystyrene sample pipe, 5min is preheated in 37 DEG C of water-baths, adding 10 μ L EDTA-2Na makes
With liquid, 10 μ L SYBR Green Ι standard solutions are rapidly added after mixing, 37 DEG C of lucifuges are incubated 10min, 4 DEG C of 3000r
min-15min is centrifuged, abandons supernatant (removing culture medium and other impurities), PBS is centrifuged again after redissolving.Above-mentioned centrifugally operated is repeated,
Supernatant is abandoned, it is stand-by that 10 μ L PBS redissolve precipitation.
4. the preparation of immune magnetic Nano material
200 μ L carboxyls magnetic beads (carboxyl magnetic bead concentration is 1mg/mL) are taken, ultrasonic 30s, Magneto separate, one are cleaned with distilled water
It is secondary.250 μ L EDC/NHS solution are added, 30min is activated at ambient temperature, Magneto separate, is cleaned repeatedly with PBS solution 3 times,
Produce magnetic bead after activating.
150 μ L are added in magnetic bead after activation resists anti-salmonella typhimurium antibody (to be diluted to 10 μ gmL-1), room temperature
Under the conditions of react 6h, Magneto separate, use 0.01molL-1PBS solution is cleaned 3 times repeatedly, obtains immune magnetic Nano material.
5. the capture and detection of salmonella typhimurium
Immune magnetic Nano material is added in the culture medium for having salmonella typhimurium, is incubated at room temperature 1h, is captured
Salmonella typhimurium be trapped by membrane filtration operation on filter membrane, filter membrane is put on Magneto separate frame clear with PBS solution
Wash the impurity for further removing and being trapped on filter membrane.Filter membrane after said process is irradiated with laser, the temperature after pre-irradiation
Change is recorded using thermal imaging system.Standard curve is established using temperature increase (Δ T) and cell number, calculation formula is such as
Under：
Δ T=Δ T1- Δs T0
Δ T, temperature increase；
Δ T0, the temperature increase after blank (being not added with cell) sample laser pre-irradiation；
Δ T1, add the temperature increase after laser pre-irradiation after sample.
In order to eliminate the influence for being trapped in unnecessary immune magnetic Nano material on filter membrane, blank (should be not added with mouse typhus
Salmonella) temperature increase (Δ T0) after sample laser pre-irradiation is from the temperature liter after laser pre-irradiation after addition sample
Deducted in high level (Δ T1).
As a result with discussion
1. the optimization of carboxyl magnetic bead
In order to select optimal carboxyl magnetic bead parameter, with laser irradiate various concentrations different-grain diameter carboxyl magnetic bead and GOs simultaneously
Its temperature variations is recorded, compares its photo-thermal effect；The carboxyl magnetic bead of same concentrations different-grain diameter is entered using Magneto separate frame
Row Magneto separate, compare its Magneto separate time；And incubated with the carboxyl magnetic bead of same concentrations different-grain diameter with salmonella typhimurium
Educate, compare its non-specific adsorption ability to bacterium, in summary select carboxyl magnetic bead at 3 points.As shown in Fig. 2
Particle diameter is 100~200nm and the photo-thermal effect and GOs of 200~300nm carboxyl magnetic beads are almost suitable, and particle diameter is 10nm carboxyl
Magnetic bead photo-thermal effect is relatively weak.Because the operating time of the Magneto separate in detection process can produce to final total operating time
Influence, as can be known from Fig. 3, particle diameter is that 100~200nm and 200~300nm carboxyls magnetic bead can complete Magneto separate in 5 s, and grain
Footpath is that can not still complete Magneto separate in 10nm carboxyl magnetic bead 5min.As shown in figure 4, particle diameter is 10nm and 100~200nm carboxylics
Base magnetic bead is higher to the non-specific adsorption of bacterium, and particle diameter is that 200~300nm carboxyls magnetic bead is relatively low to the non-specific adsorption of bacterium.
In summary, particle diameter is selected to be used for the foundation of final detection method for 200~300nm carboxyls magnetic bead.
2. the sign of immune magnetic Nano material identification salmonella typhimurium
Immune magnetic Nano material identification salmonella typhimurium is characterized using TEM and flow cytometer.Such as Fig. 5
It is shown, salmonella typhimurium is divided into the middle part of great circle, it is 200~300nm immune magnetic Nano materials to be divided into particle diameter in the middle part of ringlet
Material, as can be seen from the figure immune magnetic Nano material is successfully coupled to salmonella typhimurium surface.The application further uses
Flow cytometer is characterized to immune magnetic Nano material identification salmonella typhimurium, using with green fluorescence
SYBR Green Ι dye to salmonella typhimurium, using the anti-salmonella typhimurium antibody of Cy5 marks with being immunized
Magnetic Nano material is incubated.As shown in Figure 6, it is known that rubescent color fluorescence and green fluorescence respectively with the bacterium pair in light field
Should be consistent, it is also corresponding consistent after red fluorescence is merged with green fluorescence, it is anti-further to prove that coupling has Cy5 to mark
The salmonella typhimurium of the success of carboxyl magnetic bead and SYBR Green Ι dyeing of salmonella typhimurium antibody is coupled.
3. the optimization of laser power and irradiation time
5 μ L carboxyl magnetic bead 2min are irradiated with the 808nm laser of different capacity, utilize carboxyl after thermal imaging system record pre-irradiation
The temperature variations of magnetic bead.When irradiation power is less than 2.5Wcm-2When, with the increase of irradiation power, the temperature of carboxyl magnetic bead
Lift-off value has obvious increase, when irradiation power is in 2.5Wcm-2~2.8Wcm-2When, the temperature increase of carboxyl magnetic bead
Have no and dramatically increase (Fig. 7 A).If irradiation power is more than 2.8Wcm-2, the heating of carboxyl magnetic bead is too high then can be broken to filter membrane generation
It is bad.Therefore, laser irradiation power selection is 2.5Wcm-2.In order to realize quick detection, irradiation time should shorten as far as possible, because
Irradiation time is optimized for this.It is 2.5Wcm with irradiation power-2808nm laser irradiate 5 μ L carboxyl magnetic bead, profit
The temperature variations of carboxyl magnetic bead after pre-irradiation are recorded with thermal imaging system.As shown in Fig. 7 B, carboxyl magnetic bead is before laser irradiation
2min is brought rapidly up, slow from 2min~5min heatings.Therefore, irradiation time selection is 2min.
4. the optimization of immune nano material addition
In order to optimize the addition of immune nano material, fixation of bacteria number (1000) is received with different number of be immunized
Rice material is incubated, and after membrane filtration, Magneto separate, (808nm, 2.5Wcm are irradiated with laser-2).Fig. 8 shows, when immune
In 5~10 μ g, with the increase of immune nano material addition, photo-thermal effect increases nano material addition therewith, in addition
To reach maximum during 10 μ g, compound addition continues to increase (10~12 μ g), and photo-thermal effect drastically reduces, and this is probably
Caused by being reunited due to immune magnetic Nano material on filter membrane.Therefore, immune nano material addition selection is 10 μ
G/1000 bacterium.
The detection of salmonella typhimurium in 5.PBS solution
Because PBS matrix is smaller on testing result influence, therefore detection is carried out first in PBS.Filter membrane is irradiated with laser
It is upper to pass through filtration fraction, record temperature variations using thermal imaging system, using the photo-thermal effect of carboxyl magnetic bead establish Δ T with it is thin
Standard curve between bacterium number mesh.
It is minimum detection limit according to 3 times of levels of noise.Under the same conditions, 10 filter membrane 2min, 10 filters are irradiated with laser
The SD of temperature difference is 0.44 DEG C, the as level of noise of detection method after film pre-irradiation.Therefore, mouse typhus in corresponding standard curve
The Δ T of salmonella minimum detection limit should be 1.32 DEG C.From Fig. 9 A, based on immune magnetic Nano material photo-thermal effect
Salmonella typhimurium method lowest detection is limited to 300 bacteriums, and temperature increase Δ T and number of bacteria are 300~1000
In the range of linear dependency relation.
6. the detection of salmonella typhimurium in drinking water
In order to investigate the salmonella typhimurium method based on immune magnetic Nano material photo-thermal effect of foundation in reality
Feasibility in sample, salmonella typhimurium is added in drinking water, and is detected with method for building up.By different numbers
Purpose salmonella typhimurium is added in 1mL drinking water, and sample is after incubation, membrane filtration, Magneto separate, laser irradiation, root
Detected according to photo-thermal effect, standard curve is established according to temperature increase Δ T and salmonella typhimurium number.Fig. 9 A tables
Bright, the standard curve established in drinking water and the standard curve established in PBS are basically identical, it was demonstrated that drink water-based pair
Detection method influences smaller, temperature increase Δ T and the number of bacteria also linear dependency relation in the range of 300~1000.Sample
Product addition recovery experiment shows that the rate of recovery is 96.2%~106.4% (Fig. 9 B), can meet testing requirements.300 incubations have
The salmonella typhimurium of immune magnetic Nano material in drinking water through laser irradiate caused by temperature increase be 1.17
DEG C, the rate of recovery 96.2%, with PBS testing result it is basically identical, meet testing requirements (Fig. 9 C).
7. method specificity is investigated
In order to investigate, method for building up is specific and selective, chooses Escherichia coli, staphylococcus aureus to method
Investigated.As shown in Figure 9 C, following experiment has been carried out in PBS, salmonella typhimurium is only added in PBS, is only added
Enterobacteria is increased, only adds staphylococcus aureus.It is seen that only Escherichia coli and golden yellow grape are added in addition
Coccus group through laser irradiate caused by Δ T to be less than other two groups of experiments.More than experiment prove that the present invention establishes based on immune
The salmonella typhimurium method of magnetic Nano material photo-thermal effect has high specific and selectivity.
8. method bactericidal effect is investigated
In order to investigate the bactericidal effect of method, sample is swashed using 808nm respectively after incubation, membrane filtration, Magneto separate
Light irradiation 0s, 30s, 60s, 120s, filter membrane is rinsed repeatedly with 100 μ L PBS, 100 μ L PBS of flushing are uniform
It is applied on beef-protein medium, as shown in Figure 10, Salmonella typhimurium after 120s is irradiated with laser on filter membrane
Bacterium has been entirely killed, it was demonstrated that this method bactericidal effect is good, and the inactivation to sample can be realized while detection.
The preferred embodiment of the application is the foregoing is only, is not limited to the application, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made it is any
Modification, equivalent substitution, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. application of the immune magnetic Nano material in salmonella typhimurium quick detection.
2. application as claimed in claim 1, it is characterised in that the preparation method of the immune magnetic Nano material is：
1) carboxyl magnetic bead is taken, ultrasound, Magneto separate, is cleaned with distilled water；EDC/NHS solution is added thereto, at ambient temperature
Activation, then Magneto separate is carried out, magnetic bead after must being activated after being cleaned repeatedly with PBS solution；
2) anti-salmonella typhimurium antibody is added into the magnetic bead after activation, is reacted a period of time under room temperature condition, Magneto separate,
Then cleaned repeatedly with PBS solution, obtain immune magnetic Nano material.
3. application as claimed in claim 2, it is characterised in that the particle diameter of the carboxyl magnetic bead is that 10~300nm (is preferably 200
~300nm).
4. a kind of salmonella typhimurium quick determination method based on immune magnetic Nano material photo-thermal effect, its feature exist
In, including：
S1. carboxyl magnetic bead and anti-salmonella typhimurium antibody are coupled to obtain immune magnetic Nano material；
S2. immune magnetic Nano material step S1. being prepared is added in salmonella typhimurium, is incubated, membrane filtration,
And be further purified with magnetic separation technique, obtain salmonella typhimurium-immune magnetic Nano material composite；
S3. salmonella typhimurium-immune magnetic Nano the material composite being trapped on film is irradiated with laser, produces temperature
Change, standard curve is established according to temperature increase and number of bacteria；Photo-thermal effect based on immune magnetic Nano material simultaneously,
It is synchronous that salmonella typhimurium is inactivated.
5. detection method as claimed in claim 4, it is characterised in that the specific side of preparation of step S1. immune magnetic Nanos material
Method is：
S1.1 takes carboxyl magnetic bead, ultrasound, Magneto separate, is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature condition
Lower activation, then Magneto separate is carried out, magnetic bead after must being activated after being cleaned repeatedly with PBS solution；
S1.2 adds anti-salmonella typhimurium antibody into the magnetic bead after activation, is reacted a period of time under room temperature condition, magnetic point
From then being cleaned repeatedly with PBS solution, obtain immune magnetic Nano material.
6. detection method as claimed in claim 5, it is characterised in that the particle diameter of carboxyl magnetic bead described in step S1.1 be 10~
300nm (being preferably 200~300nm).
7. detection method as claimed in claim 4, it is characterised in that the addition of immune magnetic Nano material in step S2.
For 9~10 μ g/1000 salmonella typhimuriums (being preferably 10 μ g/1000 salmonella typhimuriums).
8. detection method as claimed in claim 4, it is characterised in that laser power is 2.5~2.8W in the step S3.
cm-2(preferably 2.5Wcm-2), wavelength 808nm, irradiation time is 2~5 minutes (being preferably 2 minutes).
9. detection method as claimed in claim 4, it is characterised in that according to temperature increase and bacterium in the step S3.
It is as follows that number establishes the specifically used formula of standard curve：
Δ T=Δ T1- Δs T0
Wherein, Δ T is temperature increase；
Δ T0 is the temperature increase after blank (being not added with salmonella typhimurium) sample laser pre-irradiation；
Δ T1 is the temperature increase after laser pre-irradiation after addition sample.
10. application of any one of the claim 4-9 detection methods in quick detection salmonella typhimurium, it is preferred that
The application mode is that salmonella typhimurium in drinking water is used for quickly detecting.
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