CN107677817B - A kind of salmonella typhimurium rapid detection method based on immune magnetic Nano material photo-thermal effect - Google Patents
A kind of salmonella typhimurium rapid detection method based on immune magnetic Nano material photo-thermal effect Download PDFInfo
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Abstract
The present invention establishes a kind of salmonella typhimurium rapid detection 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, the temperature increase that sample generates can be brought into standard curve and obtain the number of bacteria in sample.Simultaneously, utilize the magnetism and photo-thermal effect of immune magnetic Nano material, it realizes concentration and separation-detection-sample and inactivates effectively integrating for three links, this method is sensitive, safe, quick, portable, at low cost, 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 the number of bacteria linear correlativity in 300~1000 ranges, therefore the present invention has good actual application prospect, can be applied to the practical quickly detection to salmonella typhimurium.
Description
Technical field
It is the present invention relates to technical field of immunoassay, in particular to a kind of based on immune magnetic Nano material photo-thermal effect
Salmonella typhimurium rapid detection method.
Background technique
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 one of the core of control and difficult point.It is total that salmonellosis is important people and animals
One of illness, cause of disease Salmonella enteric bacteria section, can cause to poison by food.
The detection method of current salmonella is generally both needed to amplification step, with increase cause of disease can recall rate, it is time-consuming and laborious,
Need could complete within 4~7 days, it can not timely, Fast Evaluation Salmonella in Food content.It is anti-currently with thallus or flagellum
The immunological method of former specific antibody is the main direction of studying of rapid detection method.The salmonella having built up is immune
Learning detection method has enzyme-linked immunization, 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, is not necessarily to
Large-scale instrument is suitable for qualitative or semi-quantitative analysis.Since most samples to be tested are feminine gender, for the quick fixed of large sample size
Property or sxemiquantitative screening can greatly improve detection efficiency, and therefore, it is quick that such rapid detection method is highly suitable for large sample size
Screening.But since such method mostly uses greatly visual or simple detection device, sensitivity is still relatively low, is only improved spirit
Sensitivity, then be equipped with and just can be suitably used for salmonella in the food samples of complex matrices for the pre-treatment step of enrichment purification
Rapid screening.
As the above analysis, most pathogenic bacteria detection methods are by concentration and separation-detection-test inactivation point at present
It being carried out for three links, step is more, and time-consuming, and most reports about analysis time pertain only to detect this link at present,
And 2h is no less than the time required to inactivating entire analytic process 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 is still to be improved.In view of this, it is urgent to provide a kind of pair of salmonella spirits
Quick, safe, quick, portable, inexpensive detection method.
Summary of the invention
In view of the above shortcomings of the prior art, inventor provides a kind of mouse based on immune magnetic Nano material photo-thermal effect
Salmonella typhi rapid detection method, this method are realized to salmonella typhimurium simultaneously using immune magnetic Nano material
Temperature raising is established in capture, detection and inactivation, while the temperature change generated using laser irradiation immune magnetic Nano material
The standard curve of value and salmonella typhimurium number realizes the quantitative detection to salmonella typhimurium.
Specifically, the present invention relates to following technical schemes:
The first aspect of the invention discloses immune magnetic Nano material in salmonella typhimurium quickly detects
Using.The immune magnetic Nano material is added in salmonella typhimurium, specifically, by carboxyl magnetic bead subscript
Anti- salmonella typhimurium antibody is remembered to which immune magnetic Nano material be made, and then is coupled with salmonella typhimurium,
It realizes and salmonella typhimurium is captured.
Wherein, the immune magnetic Nano material the preparation method comprises the following steps:
1) carboxyl magnetic bead is taken, ultrasound, Magneto separate is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature item
It is activated under part, then carries 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, under room temperature reaction a period of time, magnetic
Separation, is then cleaned repeatedly with PBS solution, obtains immune magnetic Nano material.
Wherein, the partial size of the carboxyl magnetic bead is 10~300nm (preferably 200~300nm)；It is obtained under the partial size
Immune magnetic Nano material photo-thermal effect is best, while the Magneto separate time is short, and low to the non-specific adsorption of bacterium.
The second aspect of the invention discloses a kind of mouse typhus sramana based on immune magnetic Nano material photo-thermal effect
Salmonella rapid detection method, specific steps include:
S1. carboxyl magnetic bead and anti-salmonella typhimurium antibody are coupled to obtain immune magnetic Nano material；
S2. the immune magnetic Nano material that step S1. is prepared is added in salmonella typhimurium, is incubated for, film
Filtering, and be further purified with magnetic separation technique, obtain salmonella typhimurium-immune magnetic Nano material composite；
S3. the salmonella typhimurium being trapped on film-immune magnetic Nano material composite is irradiated with laser, is generated
Temperature change establishes standard curve according to temperature increase and number of bacteria；Photo-thermal based on immune magnetic Nano material simultaneously
Effect is synchronized and is inactivated to salmonella typhimurium.
Wherein, step S1. immune magnetic Nano material it is specific the preparation method comprises the following steps:
S1.1 takes carboxyl magnetic bead, and 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 activating after being cleaned repeatedly with PBS solution；
Anti- salmonella typhimurium antibody is added into the magnetic bead after activation by S1.2, under room temperature reaction a period of time,
Then Magneto separate is cleaned repeatedly with PBS solution, obtain immune magnetic Nano material.
Wherein, the partial size of carboxyl magnetic bead described in step S1.1 is 10~300nm (preferably 200~300nm)；The grain
The immune magnetic Nano material photo-thermal effect obtained under diameter is best, while the Magneto separate time is short, and to the non-specific adsorption of bacterium
It is low；
In step S2. the additive amount 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 additive amount is too low or excessively high, will lead to photo-thermal
Effect is lower, influences experimental result；
Laser power is 2.5~2.8Wcm in the step S3.-2(preferably 2.5Wcm-2), wavelength 808nm shines
Penetrating the time is 2~5 minutes (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 excessively high, then immune magnetic Nano material warms are excessively high to generate 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- Δ T0
Wherein, Δ T is temperature increase
Δ T0 is the temperature increase before and after blank (being not added with salmonella typhimurium) sample laser irradiation.
Δ T1 is the temperature increase after adding sample before and after laser irradiation.
In order to eliminate the influence for being trapped in extra immune magnetic Nano material on filter membrane, therefore blank (should be not added with mouse wound
Cold salmonella) temperature increase (Δ T0) before and after sample laser irradiation is from the temperature before and after laser irradiation after addition sample
It is deducted in lift-off value (Δ T1)；
The invention also discloses application of the above-mentioned detection method in quickly detection salmonella typhimurium, specifically, institute
Stating application mode is 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 based on immune magnetic Nano material photo-thermal effect and quickly detects
Method is established between temperature increase and salmonella typhimurium number using the photo-thermal effect of immune magnetic Nano material
Standard curve can bring the temperature increase that sample generates into standard curve and obtain the number of bacteria in sample.Meanwhile it utilizing
The magnetism and photo-thermal effect of immune magnetic Nano material realize concentration and separation-detection-sample and inactivate the effective of three links
Integration realizes that the photo-thermal of the magnetic enrichments of pathogenic bacteria, Opto-thertnal detection and sample inactivates three kinds of function using a kind of nano material simultaneously
Can, the bulk analysis time is foreshortened within 1.5h；
This method is sensitive, safe, quick, portable, at low cost, experiments verify that, it is 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 correlativity in~1000 ranges, therefore the present invention has good actual application prospect, can be applied to mouse typhus
The practical quickly detection of salmonella.
Detailed description of the invention
Figure 1A is immune magnetic Nano material identification salmonella typhimurium schematic diagram；Figure 1B is based on immune magnetic Nano
The salmonella typhimurium of material photo-thermal effect captures 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 concentration；
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 power and carboxyl magnetic bead heat up relational graph, Fig. 7 (B) difference laser irradiation time with
Carboxyl magnetic bead heating relational graph；
Fig. 8 is immune magnetic Nano material additive amount and its photo-thermal effect relational graph；
Fig. 9 (A) is the canonical plotting of Δ T and salmonella number in PBS and drinking water, and Fig. 9 (B) is in drinking water
The testing result figure of salmonella；Fig. 9 (C) is that method specificity of the invention investigates figure, is directed to salmonella, large intestine respectively
Bacillus and staphylococcus aureus are investigated.
Figure 10 is to be incubated for carboxyl magnetic bead after laser difference irradiation time to the bactericidal effect figure of salmonella typhimurium.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has logical with 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 specific 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 singular shape
Formula be also intended to include plural form, additionally, it should be understood that, when in the present specification use term "comprising" and/or
When " comprising ", existing characteristics, step, operation, device, component and/or their combination are indicated.
As described in background technique, most pathogenic bacteria detection methods are that concentration and separation-detection-test is gone out
Living to be divided into three links progress, step is more, and time-consuming.
In view of this, being provided a kind of based on immune magnetic Nano material photo-thermal in a kind of specific embodiment of the invention
The salmonella typhimurium rapid detection method of effect, specific steps include:
S1. carboxyl magnetic bead and anti-salmonella typhimurium antibody are coupled to obtain immune magnetic Nano material；
S2. the immune magnetic Nano material that step S1. is prepared is added in salmonella typhimurium, is incubated for, film
Filtering, and be further purified with magnetic separation technique, obtain salmonella typhimurium-immune magnetic Nano material composite；
S3. the salmonella typhimurium being trapped on film-immune magnetic Nano material composite is irradiated with laser, is generated
Temperature change establishes standard curve according to temperature increase and number of bacteria；Photo-thermal based on immune magnetic Nano material simultaneously
Effect is synchronized and is inactivated to salmonella typhimurium.
Wherein, step S1. immune magnetic Nano material it is specific the preparation method comprises the following steps:
S1.1 takes carboxyl magnetic bead, and 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 activating after being cleaned repeatedly with PBS solution；
Anti- salmonella typhimurium antibody is added into the magnetic bead after activation by S1.2, under room temperature reaction a period of time,
Then Magneto separate is cleaned repeatedly with PBS solution, obtain immune magnetic Nano material.
Wherein, the partial size of carboxyl magnetic bead described in step S1.1 is 10~300nm (preferably 200~300nm)；The grain
The immune magnetic Nano material photo-thermal effect obtained under diameter is best, while the Magneto separate time is short, and to the non-specific adsorption of bacterium
It is low；
In step S2. the additive amount 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 additive amount is too low or excessively high, will lead to photo-thermal
Effect is lower, influences experimental result；
Laser power is 2.5~2.8Wcm in the step S3.-2(preferably 2.5Wcm-2), wavelength 808nm shines
Penetrating the time is 2~5 minutes (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 excessively high, then immune magnetic Nano material warms are excessively high to generate 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- Δ T0
Wherein, Δ T is temperature increase
Δ T0 is the temperature increase before and after blank (being not added with salmonella typhimurium) sample laser irradiation.
Δ T1 is the temperature increase after adding sample before and after laser irradiation.
In order to eliminate the influence for being trapped in extra immune magnetic Nano material on filter membrane, therefore blank (should be not added with mouse wound
Cold salmonella) temperature increase (Δ T0) before and after sample laser irradiation is from the temperature before and after laser irradiation after addition sample
It is deducted in lift-off value (Δ T1).
Mode by the following examples is further elaborated the present invention.
Embodiment
1. test material and equipment
1.1 main materials and reagent
1 reagent of table and drug
1.2 key instrument and equipment
Table 3-2 instrument and equipment
Table 3-2Equipments and Instruments
2 related solutions are prepared
EDC/NHS solution is prepared: weighing EDC 9.6mg respectively, NHS 9.6mg is dissolved in 3mL secondary water.
0.01mol·L-1Phosphate buffer solution is prepared: weighing Na respectively2HPO4·12H2O 13.76g、NaH2PO4·2
H2O 1.79g, NaCl 9.00g, add deionized water to be settled to 1000mL.
SYBR Green Ι standard solution is prepared: SYBR Green Ι stoste being diluted 100 times with DMSO, -20 DEG C of guarantors
It deposits.
EDTA-2Na is prepared using liquid: weighing EDTA-2Na 18.61g, sterile purified water is added to be settled to 100mL.
LB liquid medium is prepared: being weighed tryptone 10g, yeast extract 5g, NaCl 10g respectively, is used deionized water
It is settled to 1000mL, adjusts pH to 7.0.
Beef-protein medium is prepared: being weighed beef extract 3g, peptone 10g, NaCl 5g respectively, is used deionized water
It is settled to 1000mL, 20g agar is added.
Culture, preparation and the fluorescent staining method of 3 salmonella typhimuriums
The culture of 3.1 salmonella typhimuriums
It is placed in LB liquid medium with oese from picking salmonella typhimurium single colonie on inclined-plane, 37 DEG C of oscillation 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 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 bacterium solution 1mL, 4 DEG C of 3000rmin are taken respectively-1Be centrifuged 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 precipitating.
The fluorescent staining method of 3.3 salmonella typhimuriums
It takes 1mL bacterium solution in polystyrene sample pipe, 5min is preheated in 37 DEG C of water-baths, 10 μ L EDTA-2Na, which are added, to be made
With liquid, 10 μ L SYBR Green Ι standard solutions are rapidly added after mixing, 37 DEG C are protected from light incubation 10min, 4 DEG C of 3000r
min-1It is centrifuged 5min, 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 precipitating.
4. the preparation of immune magnetic Nano material
200 μ L carboxyl magnetic beads (carboxyl magnetic bead concentration is 1mg/mL) is taken, ultrasonic 30s, Magneto separate cleans one with distilled water
It is secondary.250 μ L EDC/NHS solution are added, activate 30min at room temperature, Magneto separate is cleaned 3 times repeatedly with PBS solution,
Up to magnetic bead after activation.
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 uses 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, 1h is incubated at room temperature, is captured
Salmonella typhimurium be trapped on filter membrane by membrane filtration operation, filter membrane is put on Magneto separate frame clear with PBS solution
Wash the impurity for further removing and being trapped on filter membrane.With filter membrane of the laser irradiation after the above process, the temperature of front and back is irradiated
Variation 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- Δ T0
Δ T, temperature increase；
Δ T0, the temperature increase before and after blank (being not added with cell) sample laser irradiation；
Δ T1 adds the temperature increase after sample before and after laser irradiation.
In order to eliminate the influence for being trapped in extra immune magnetic Nano material on filter membrane, blank (should be not added with mouse typhus
Salmonella) temperature increase (Δ T0) before and after sample laser irradiation is from the temperature liter before and after laser irradiation after addition sample
It is deducted in high level (Δ T1).
Results and discussion
1. the optimization of carboxyl magnetic bead
In order to select optimal carboxyl magnetic bead parameter, simultaneously with the carboxyl magnetic bead and GOs of laser irradiation various concentration different-grain diameter
Its temperature variations is recorded, its photo-thermal effect is compared；Using Magneto separate frame to the carboxyl magnetic bead of same concentrations different-grain diameter into
Row Magneto separate compares its Magneto separate time；And it is incubated with the carboxyl magnetic bead of same concentrations different-grain diameter with salmonella typhimurium
It educates, compares its non-specific adsorption ability to bacterium, in summary 3 points carboxyl magnetic bead is selected.As shown in Fig. 2,
Partial size is 100~200nm and the photo-thermal effect and GOs of 200~300nm carboxyl magnetic bead are almost suitable, and partial size is the carboxyl of 10nm
Magnetic bead photo-thermal effect is relatively weak.Since the operating time of Magneto separate in the detection process can generate final total operating time
It influences, as can be known from Fig. 3, partial size is that 100~200nm and 200~300nm carboxyl magnetic bead can complete Magneto separate in 5 s, and grain
Magneto separate is still unable to complete in the carboxyl magnetic bead 5min that diameter is 10nm.As shown in figure 4, partial size is 10nm and 100~200nm carboxylic
Base magnetic bead is higher to the non-specific adsorption of bacterium, and partial size is that 200~300nm carboxyl magnetic bead is lower to the non-specific adsorption of bacterium.
In conclusion selecting foundation of the partial size for 200~300nm carboxyl magnetic bead for final detection method.
2. the characterization 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 material that partial size is divided into 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 characterizes immune magnetic Nano material identification salmonella typhimurium, using with green fluorescence
SYBR Green Ι dyes salmonella typhimurium, using the Cy5 anti-salmonella typhimurium antibody marked and is immunized
Magnetic Nano material is incubated for.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 carboxyl magnetic bead success of salmonella typhimurium antibody and the salmonella typhimurium of SYBR Green Ι dyeing are coupled.
3. the optimization of laser power and irradiation time
With the 5 μ L carboxyl magnetic bead 2min of 808nm laser irradiation of different capacity, irradiation front and back carboxyl is recorded using thermal imaging system
The temperature variations of magnetic bead.When irradiation power is lower than 2.5Wcm-2When, with the increase of irradiation power, the temperature of carboxyl magnetic bead
Lift-off value has apparent increase, when irradiation power is in 2.5Wcm-2~2.8Wcm-2When, the temperature increase of carboxyl magnetic bead
It has no and dramatically increases (Fig. 7 A).If irradiation power is greater than 2.8Wcm-2, the heating of carboxyl magnetic bead is excessively 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 in this.It is 2.5Wcm with irradiation power-25 μ L of 808nm laser irradiation carboxyl magnetic bead, benefit
The temperature variations of irradiation front and back carboxyl magnetic bead 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 heating.Therefore, irradiation time is selected as 2min.
4. the optimization of immune nano material additive amount
In order to optimize the additive amount of immune nano material, fixation of bacteria number (1000) is received with different number of be immunized
Rice material is incubated for, and after film filtering, Magneto separate, is irradiated (808nm, 2.5Wcm with laser-2).Fig. 8 shows when immune
Nano material additive amount increases, photo-thermal effect increases therewith, in additive amount in 5~10 μ g with immune nano material additive amount
To reach maximum value when 10 μ g, compound additive amount continues growing (10~12 μ g), and photo-thermal effect drastically reduces, this may be
Caused by being reunited on filter membrane as immune magnetic Nano material.Therefore, immune nano material additive amount is selected as 10 μ
G/1000 bacterium.
The detection of salmonella typhimurium in 5.PBS solution
Since PBS matrix is smaller on testing result influence, detection carries out first in PBS.With laser irradiation filter membrane
It is upper to pass through filtration fraction, temperature variations are recorded using thermal imaging system, establish Δ T and thin using the photo-thermal effect of carboxyl magnetic bead
Standard curve between bacterium number mesh.
It is minimum detection limit according to 3 times of levels of noise.Under the same conditions, with 10 filter membrane 2min of laser irradiation, 10 filters
The SD of film irradiation front and back temperature difference is 0.44 DEG C, the as level of noise of detection method.Therefore, mouse typhus in corresponding standard curve
The Δ T of salmonella minimum detection limit should be 1.32 DEG C.By Fig. 9 A it is found that 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
Linear correlativity in range.
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 by being incubated for, film filters, after Magneto separate, laser irradiation, root
It is detected according to photo-thermal effect, standard curve is established according to temperature increase Δ T and salmonella typhimurium number.Fig. 9 A table
Bright, the standard curve established in drinking water and the standard curve established in PBS are almost the same, it was demonstrated that drink water-based pair
Detection method influences smaller, temperature increase Δ T and the number of bacteria also linear correlativity in 300~1000 ranges.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 temperature increase that the salmonella typhimurium of immune magnetic Nano material generates in drinking water through laser irradiation is 1.17
DEG C, the rate of recovery 96.2%, in PBS testing result it is almost the same, meet testing requirements (Fig. 9 C).
7. method specificity is investigated
The specificity and selectivity of method for building up in order to investigate choose Escherichia coli, staphylococcus aureus to method
It is investigated.As shown in Figure 9 C, following experiment has been carried out in PBS, and salmonella typhimurium is only added in PBS, is only added
Enterobacteria is increased, staphylococcus aureus is only added.It is seen that only Escherichia coli and golden yellow grape are added in addition
The Δ T that coccus group is generated through laser irradiation will be lower than other two groups of experiments.Above experiments have shown that the present invention establish 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 by being incubated for, film filters, after Magneto separate, is swashed respectively using 808nm
Light irradiates 0s, 30s, 60s, 120s, carries out repeated flushing to filter membrane with 100 μ L PBS, and 100 μ L PBS of flushing are uniform
It is applied on beef-protein medium, as shown in Figure 10, Salmonella typhimurium after with laser irradiation 120s 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 foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any
Modification, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (6)
1. a kind of salmonella typhimurium rapid detection method based on immune magnetic Nano material photo-thermal effect, feature exist
In, comprising:
S1. carboxyl magnetic bead and anti-salmonella typhimurium antibody are coupled to obtain immune magnetic Nano material；
Step S1. immune magnetic Nano material it is specific the preparation method comprises the following steps:
It is the carboxyl magnetic bead of 200~300nm that S1.1, which takes partial size, and ultrasonic, Magneto separate is cleaned with distilled water；EDC/ is added thereto
NHS solution, activates at room temperature, then carries out Magneto separate, magnetic bead after must activating after being cleaned repeatedly with PBS solution；
Anti- salmonella typhimurium antibody is added into the magnetic bead after activation by S1.2, under room temperature reaction a period of time, magnetic point
From then being cleaned repeatedly with PBS solution, obtain immune magnetic Nano material；
S2. the immune magnetic Nano material that step S1. is prepared is added in salmonella typhimurium, is incubated for, film filtering,
And be further purified with magnetic separation technique, obtain salmonella typhimurium-immune magnetic Nano material composite；
The additive amount of immune magnetic Nano material is 9~10 μ g/1000 salmonella typhimuriums in step S2.；
S3. the salmonella typhimurium being trapped on film-immune magnetic Nano material composite is irradiated with laser, generates temperature
Variation, establishes standard curve according to temperature increase and number of bacteria；Photo-thermal effect based on immune magnetic Nano material simultaneously,
It synchronizes and salmonella typhimurium is inactivated；
Laser power is 2.5~2.8Wcm in step S3.-2, wavelength 808nm, irradiation time is 2~5 minutes.
2. detection method as described in claim 1, which is characterized in that the additive amount of immune magnetic Nano material in step S2.
For 10 μ g/1000 salmonella typhimuriums.
3. detection method as described in claim 1, which is characterized in that laser power is 2.5Wcm in the step S3.-2,
Irradiation time is 2 minutes.
4. detection method as described in claim 1, which is characterized 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- Δ T0
Wherein, Δ T is temperature increase；
Δ T0 is the temperature increase before and after blank sample laser irradiation；
Δ T1 is the temperature increase after adding sample before and after laser irradiation.
5. application of any one of the claim 1-4 detection method in quickly detection salmonella typhimurium.
6. as claimed in claim 5 application, which is characterized in that the application mode be to salmonella typhimurium in drinking water into
Row quickly detection.
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