CN109060178A - A kind of collection detection carrier and signal result from integrated thermometer rapid detection method - Google Patents
A kind of collection detection carrier and signal result from integrated thermometer rapid detection method Download PDFInfo
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Abstract
The invention discloses a kind of, and the collection detection carrier based on graphene oxide photo-thermal effect results from integrated thermometer detection method with signal, and using thermometer as carrier, the characteristic based on nano material sets up sensitive, the quick method for detecting target determinand.Thermometer is as detection method carrier, the temperature change that can be generated simultaneously to photo-thermal effect is read, and is simplified the operating procedure of entire method, is significantly reduced testing cost, and detection sensitivity is substantially better than immunity test strip, facilitates the extensive use of detection method.
Description
Technical field
The present invention relates to a kind of collection detection carriers and signal to result from integrated thermometer rapid detection method, belongs to detection
Technical field.
Background technique
The rapid detection method for establishing a kind of easy, sensitive food hazardous material is the effective way to ensure food safety.
The rapid detection method for hazardous material in food established at present is mostly using ELISA Plate, test strips etc. as carrier, including enzyme
Linked immunosorbent assay, immunity test strip and immunosensor etc..Enzyme-linked immunization sensitivity is relatively high but complicated for operation, and needs enzyme
It marks instrument and is not suitable for field quick detection.Test strips method is easy to operate, cost is relatively low, and range estimation or card reader etc. can be used and simply examine
Measurement equipment obtains qualitative or semi-quantitative analysis, but the detection method sensitivity is relatively low.In addition, the common carrier nitre of test strips
The constituent of acid cellulose film is complicated, and the combination of the albumen such as film and antibody is affected by many factors, while in operation
The reasons such as frangible, easy to break and generation scratch keep the sensitivity of test strips and stability all to be improved.Therefore it finds and stablizes, is suitable
The detection carrier of operation becomes the research hotspot of researcher.
Photo-thermal effect is mainly used in photo-thermal therapy and bio-imaging of cancer etc. at present, directly answers photo-thermal effect
Research for establishing detection method is less.The thermal lensing effect that laser irradiation generates is used for heat tracking by Dovichi et al.
Analysis is earliest to be applied to photo-thermal effect to establish the research of detection method at present.Qin et al. is imitated using the photo-thermal of colloidal gold
It answers, establishes the standard curve of temperature increase and testing concentration, compared with test strips estimate detection method, the inspection of this method
It surveys sensitivity and improves 32 times.Photo-thermal effect is applied to detection 2,4,6-trinitrotoluene by Huang et al., and detection limit is reachable
14ng/cm.In the research work before this seminar: Zhou et al. establishes a kind of based on nanocomposite photo-thermal effect
Cancer cell percolation type test strips detection method, detection limits up to 600 cells.Zhang et al. establishes a kind of based on oxygen
The circulating tumor cell detection method of graphite olefinic functionality immunomagnetic beads, the temperature after establishing graphene oxide irradiation increase
Standard curve between value and the number of circulating tumor cell, detection are limited up to 100 cells.But it is above-mentioned using photo-thermal effect
The related detecting method that should be established, temperature change needs to record by thermal imaging system or infrared temperature-measuring gun is read, and increases reality
Test cost.
Diagnosis and the photo-thermal therapy that there is optical-thermal conversion material photo-thermal effect to be widely used in tumour.But detection at present
In method, the research for being directly applied to establish rapid detection method for the photo-thermal effect of optothermal material is relatively fewer, and photo-thermal
The higher cost (about 40,000 yuan) that the instrument (thermal imaging system) of temperature is detected in the related detecting method that effect is established, it is wide to limit it
General usability and popularization.Therefore, a kind of cheap, easy to operate, detection quickly collection function vector and detection function are developed
It can be extremely urgent in integrated rapid detection method.
Summary of the invention
For the technical problems in the prior art, it is an object of the present invention to provide one kind to be based on graphene oxide
The thermometer rapid detection method of photo-thermal effect.The present invention is the photo-thermal effect using thermometer as carrier, based on nano material
Sensitive, the quick target object detecting method to be measured that should be set up.Thermometer (about 60 yuan) is both used as experimental vehicle, while can be right
The temperature change that photo-thermal effect generates is read, and is simplified operating procedure, is also greatly reduced testing cost, helps to examine
The extensive use of survey method.
A second object of the present invention is to provide a kind of coupling methods of thermometer mercury head surface antibody.Target is to be measured
Object antibody modification allows to specificity capture target determinand to thermometer mercury head surface, to improve the sensitive of detection method
Degree lays the foundation.
To achieve the above object, the technical solution of the present invention is as follows:
The present invention provides a kind of coupling for collecting detection carrier and resulting from integrated thermometer mercury head surface antibody with signal
Method, comprising the following steps:
(1) thermometer surface preparation
Detergent cleaning temperature meter mercury head surface is first used, surface smut is removed, is then cleaned with deionized water, drying is extremely
After anhydrous, thermometer mercury head is placed in ultrasound in Piranha solution, is cleaned with a large amount of deionized water to mercury head surface pH
It is dry for neutrality；Restore after thermometer to room temperature, is dipped in 3- aminopropyl triethoxysilane ((3-aminopropyl)
Triethoxysilane, APTES) it reacts in methanol solution；Then thermometer mercury head is rinsed with methanol, washed away remaining
Thermometer is placed in baking oven and dries by APTES；
(2) coupling of thermometer mercury head surface antibody
Target determinand antibody is added in polystyrene sample pipe, the thermometer mercury head that step (1) is handled well is inserted
Enter and be incubated for into antibody-solutions, rinses mercury head with buffer, wash away unbonded antibody.
Preferably, in the step (1), ultrasonic time is 0.5~1.5h；Further preferably 1h.
Preferably, in the step (1), dry condition is 60~70 DEG C of dryings after the ultrasonic cleaning of thermometer mercury head
1.5~2.5h, preferably 65 DEG C dry 2h.
Preferably, in the step (1), reaction condition are as follows: room temperature reaction 2~2.5h of time；Further preferably room temperature
Reaction time 2h.
Preferably, in the step (1), thermometer is 50~60 DEG C of 25~35min of drying in baking oven drying condition；Into one
Step is preferably 50 DEG C of drying 30min.
Preferably, in the step (1), the volume of Piranha solution is 20~25mL.
Preferably, in the step (1), the mass fraction of APTES methanol solution is 10%.
Preferably, in the step (1), methanol rinse thermometer mercury head number be 2~3 times, further preferred 3
It is secondary.
Preferably, in the step (2), the dosage of antibody-solutions is 300~350 μ L, and concentration is 3 μ g/mL.
Preferably, in the step (2), incubation conditions are 35~40 DEG C of 1~2h of incubation；Further preferably 37 DEG C incubations
1.5h。
Preferably, in the step 1.2, buffer is 0.01mol/L PBS, is rinsed 2~3 times.
The present invention provide it is a kind of based on graphene oxide photo-thermal effect collection detection carrier and signal result from integrated temperature
Degree meter detection method, comprising:
(1) thermometer mercury head surface is coupled target determinand antibody
After being pre-processed to thermometer mercury head surface, the thermometer mercury head handled well is inserted into antibody-solutions
It is incubated for, cleaning；
(2) preparation of graphene is immunized
Target determinand antibody is added in graphene oxide solution and is reacted, obtains antibody-graphene compound, i.e.,
Immune graphene；
(3) detection of target determinand.
The concrete operation method of the step (1) are as follows:
1.1 thermometer surface preparations
Detergent cleaning temperature meter mercury head surface is first used, surface smut is removed, is then cleaned with deionized water, drying is extremely
After anhydrous, thermometer mercury head is placed in ultrasound in Piranha solution, is cleaned with a large amount of deionized water to mercury head surface pH
It is dry for neutrality；Restore after thermometer to room temperature, is dipped in 3- aminopropyl triethoxysilane ((3-aminopropyl)
Triethoxysilane, APTES) it reacts in methanol solution；Then thermometer mercury head is rinsed with methanol, washed away remaining
Thermometer is placed in baking oven and dries by APTES；
The coupling of 1.2 thermometer mercury head surface antibody
Target determinand antibody is added in polystyrene sample pipe, the thermometer mercury head that step (1) is handled well is inserted
Enter and be incubated for into antibody-solutions, rinses mercury head with buffer, wash away unbonded antibody.
Preferably, in the step 1.1, ultrasonic time is 0.5~1.5h；Further preferably 1h.
Preferably, in the step 1.1, dry condition is 60~70 DEG C of dryings after the ultrasonic cleaning of thermometer mercury head
1.5~2.5h, preferably 65 DEG C dry 2h.
Preferably, in the step 1.1, reaction condition are as follows: room temperature reaction 2~2.5h of time；Further preferably room temperature
Reaction time 2h.
Preferably, in the step 1.1, thermometer is 50~60 DEG C of 25~35min of drying in baking oven drying condition；Into one
Step is preferably 50 DEG C of drying 30min.
Preferably, in the step 1.1, the volume of Piranha solution is 20~25mL.
Preferably, in the step 1.1, the mass fraction of APTES methanol solution is 10%.
Preferably, in the step 1.1, methanol rinse thermometer mercury head number be 2~3 times, further preferred 3
It is secondary.
Preferably, in the step 1.2, the dosage of target determinand antibody is 300~350 μ L, and concentration is 3 μ g/mL.
Preferably, in the step 1.2, incubation conditions are 35~40 DEG C of 1~2h of incubation；Further preferably 37 DEG C incubations
1.5h。
Preferably, in the step 1.2, buffer is 0.01mol/L PBS, is rinsed 2~3 times.
In the step (2), obtained immune graphene is saved at 4 DEG C.
Preferably, the reaction condition is 5~7h of room temperature concussion reaction；Further preferably room temperature concussion reaction 6h.
Preferably, the volume ratio of the target determinand bacteria antibody and graphene oxide solution is 1:1, target determinand bacterium
Antibody concentration is 10 μ g/mL, and graphene oxide solution concentration is 200 μ g/mL.
The concrete operation method of the step (3) are as follows:
By step (1) surface modification there is target determinand antibody thermometer to be inserted into target determinand solution to incubate
It educates, buffer rinses thermometer mercury head, washes away unbonded determinand；Then the mercury head that will be captured target determinand is inserted
Enter into immune graphene solution and be incubated for, rinses thermometer with buffer；Laser irradiation mercury head position is used after its drying, is shone
The temperature change for penetrating front and back directly passes through temperature reading；Standard song is established using temperature increase and target testing concentration
Line.
Preferably, the condition being incubated in target determinand is 35~40 DEG C of 1.5~2.5h of incubation；Further preferably
37 DEG C of incubation 2h.
Preferably, the incubation conditions in immune graphene solution are 35~40 DEG C of 1.5~2.5h of incubation；It is further excellent
37 DEG C of incubation 2h are selected as,
Preferably, the buffer is 0.01mol/L PBS, is rinsed 3~4 times.
The determinand is cancer cell, bacterium, high molecular weight protein and small-molecule substance.
Preferably, the bacterium is salmonella, and high molecular weight protein is anaphylactogen and carcinomebryonic antigen, and small-molecule substance is agriculture
Residue of veterinary drug；
Preferably, graphene can be replaced with the optical-thermal conversion materials such as colloidal gold, nanoscale magnetic bead also in this hair by the present invention
Within bright protection scope.
Preferably, thermometer carrier used in the present invention can be various types of thermometers.
Preferably, the identification material in the present invention can be the materials such as antibody, molecularly imprinted polymer and aptamers.
The present invention also provides a kind of above-mentioned detection methods in detection cancer cell, bacterium, high molecular weight protein and small-molecule substance
In application.
Preferably, the bacterium is the pathogenic bacteria such as salmonella, and high molecular weight protein is anaphylactogen and carcinomebryonic antigen etc., small point
Sub- substance is agricultural and veterinary chemicals residual etc..
Thermometer detection method of the present invention detection cancer cell, bacterium, high molecular weight protein building block principle be according to dual anti-folder
Heart principle, detection small-molecule substance is according to competitive binding principle.
Beneficial effect
1, plan utilization thermometer of the present invention as carrier, set up sensitive, quick by the photo-thermal effect based on nano material
Collection detection carrier and signal result from integrated thermometer detection method.It is used for the implementation that salmonella is sensitive, quickly detects
Scheme, entire detection time are about 2h, hence it is evident that lower than the detection process about 3h of enzyme linked immunological.Its detection sensitivity is up to 103CFU/
ML, hence it is evident that better than the detection sensitivity of immunity test strip.
2, microplate reader (about 3.5 ten thousand yuan) or card reader (about 20,000 are needed for the quantitative detection of enzyme linked immunological or test strips
Member), and (about 40,000 yuan) are also reached by the thermal imaging system cost that optical-thermal conversion material performance carries out quantitative detection, the instrument of great number at
Originally the widely available of traditional fast quantitative measurement method for detecting is limited.The present invention generates photo-thermal effect using thermometer (about 60 yuan)
Temperature change read, significantly reduce testing cost, facilitate the extensive use of detection method.
3, the present invention is using thermometer as detection method carrier, while can carry out to the temperature change that photo-thermal effect generates
Reading, collection detection carrier and signal result from one, simplify the operating procedure of entire method.
4, the present invention realizes that the Photothermal Signals of graphene are converted using laser illumination, directly records temperature using thermometer
Variation, and the relationship between target determinand and temperature change is established, it realizes to the quantitative, quick of target determinand, Sensitive Detection.
5, the present invention provides a kind of coupling method of thermometer mercury head surface antibody, by glass-stem thermometer surface
Mixed and disorderly valence link is handled, make its surface have it is stable can connect target identification material (antibody, molecularly imprinted polymer or
Aptamers) carrier of the active group as detection method.
Detailed description of the invention
Fig. 1 is that the collection detection carrier and signal based on graphene oxide photo-thermal effect result from integrated Salmonella typhimurium
Bacterium capture and detection process schematic diagram.
The optimization of Fig. 2 thermometer coupled antibody concentration.
The optimization of Fig. 3 graphene coupled antibody concentration.
The standard curve of Fig. 4 Δ T and salmonella typhimurium number.
Fig. 5 method specificity is investigated.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and examples.It is tested below the present invention to detect mouse typhus sand
Detection method is illustrated for door Salmonella, but is not limited to detection salmonella typhimurium.
The coupling method of 1 thermometer mercury head surface antibody of embodiment
1, thermometer surface preparation
Detergent cleaning temperature meter mercury head surface is used first, is removed surface smut, is then cleaned with deionized water, to temperature
Degree meter mercury head surface is dry to be placed in thermometer mercury head in 20mL Piranha solution, ultrasonic 1h to after anhydrous, with a large amount of
Deionized water clean to mercury head surface pH for neutrality, 65 DEG C of dry 2h.Restore after thermometer to room temperature, is dipped in
In 10% 3- aminopropyl triethoxysilane ((3-aminopropyl) triethoxysilane, APTES) methanol solution, room
Temperature reaction 2h, is then rinsed thermometer mercury head 3 times with methanol, washes away remaining APTES, thermometer is placed in 50 in baking oven
DEG C drying 30min.
2, the coupling of thermometer mercury head surface antibody
The anti-salmonella typhimurium antibody that 300 μ L concentration are 3 μ g/mL is added in polystyrene sample pipe.It will processing
Good thermometer mercury head is inserted into anti-salmonella typhimurium antibody solution, 37 DEG C of incubation 1.5h, with 0.01mol/L PBS
It rinses mercury head 3 times, washes away the thermometer mercury head that unbonded antibody is modified to get salmonella typhimurium antibody.
3, the optimization of thermometer surface antibody modification amount
Anti- salmonella typhimurium antibody modification to thermometer mercury head surface is captured into Salmonella typhimurium with specificity
Bacterium.The amount of the antibody of mercury head surface coupling will have a direct impact on the capture rate to bacterium, thus the sensitivity to detection method
It has an impact.
FITC-IgG is modified to thermometer mercury head surface and is optimized to antibody dosage by the present invention.By various concentration
Fluorescence antibody (0.1,0.5,1,2,3,4,5 μ g/mL) and thermometer mercury head are in 37 DEG C of incubation 1.5h, with 0.01mol/L PBS
It rinses mercury head 3 times, washes away unbonded antibody, finally eluted fluorescence antibody with 1mol/L NaOH, it is glimmering by surveying its
Luminous intensity optimizes the number of thermometer surface antibody modification amount.As seen from Figure 2, glimmering with the increase of antibody concentration
Luminous intensity is also gradually increasing.When fluorescence antibody concentration is 3 μ g/mL, the fluorescence intensity of the antibody of elution nearly reaches maximum
Value, with the increase of concentration, fluorescence intensity increases unobvious.Therefore, the antibody concentration after optimization is 3 μ g/mL.
The preparation method of graphene is immunized in embodiment 2
1, the culture of salmonella typhimurium
It falls in LB liquid medium from picking salmonella typhimurium single bacterium on inclined-plane, is received after 37 DEG C of shaken cultivation 12h
Collect bacterium solution.The interference for first handling salmonella typhimurium before using bacterium solution to reduce culture medium to experiment is centrifuged in 1.5mL
A small amount of bacterium solution is added in pipe, 3000r/min is centrifuged 5min, discards supernatant liquid, then redissolved and precipitated with PBS solution, then be centrifuged, repeats
It above-mentioned steps 3 times, is dissolved again with PBS solution later and precipitates and use.
2, the preparation of graphene is immunized
The anti-salmonella typhimurium antibody that 1mL concentration is 10 μ g/mL is added to the oxidation that 1mL concentration is 200 μ g/mL
In graphene solution, room temperature concussion reaction 6h obtains the compound of antibody graphene, i.e., immune graphene, 4 DEG C of preservations.
3, the optimization of graphene coupled antibody concentration
Graphene is integrated to thermometer mercury head surface, graphene coupled antibody by the antibody capture bacterium of coupling
Amount will will affect the amount for being connected to the graphene of mercury head surface, eventually temperature rise effect is had an impact, to influence to examine
The sensitivity of survey method, it is therefore desirable to the concentration for the antibody that optimization is incubated for graphene.
It is centrifuged after the FITC-IgG of graphene and various concentration is incubated for, the fluorescence intensity by detecting the compound optimizes
The dosage of antibody.The dosage of fixed graphene (2mg/mL, 150 μ L) is constant, respectively with the FITC-IgG of various concentration (0.5,1,
5,10,20 μ g/mL) 37 DEG C be incubated for 2h after, 11200 × g is centrifuged 10min, abandons supernatant, after being resuspended with 0.01mol/L PBS again
Centrifugation repetitive operation 2 times, finally detects the fluorescence intensity of compound.As seen from Figure 3, with the increase of antibody concentration, resist
Body fluorescence intensity also gradually increases.When antibody concentration is 10 μ g/mL, fluorescence intensity nearly reaches maximum value, and with anti-
The increase of bulk concentration, fluorescence intensity increase unobvious.Therefore select 10 μ g/mL as the antibody concentration of optimization.
The capture and detection method of 3 salmonella typhimurium of embodiment
The thermometer that the surface modification of above-mentioned preparation has anti-salmonella typhimurium antibody is inserted into salmonella solution
In, 37 DEG C of incubation 2h, 0.01mol/L PBS are rinsed thermometer 3 times, wash away unbonded bacterium.Then it will be captured mouse typhus
The mercury head of salmonella is rinsed thermometer 3 times after being inserted into 37 DEG C of incubation 2h with PBS.Laser irradiation mercury is used after its drying
Head position, the temperature change for irradiating front and back directly pass through temperature reading.Utilize temperature increase (Δ T, formula 1) and bacterial population
Mesh establishes standard curve.
Δ T=Δ T1- Δ T0 formula 1
Δ T, temperature increase
Δ T0, the temperature increase before and after blank (being not added with bacterium) sample laser irradiation.
Δ T1 adds the temperature increase after sample before and after laser irradiation.
In order to eliminate non-specific adsorption to mercury head surface graphene influence, should be by blank (being not added with bacterium) sample
Temperature increase (Δ T0) before and after product laser irradiation is from the temperature increase (Δ T1) before and after laser irradiation after addition sample
It deducts.
With laser irradiation thermometer mercury head point, temperature change is obtained by temperature reading, utilizes the light of graphene
Fuel factor establishes the relationship between temperature increase △ T and bacterial number.As shown in Figure 4, the concentration of salmonella typhimurium exists
103~108It is in good linear relationship within the scope of CFU/mL, the lowest detection for the detection method established is limited to 103CFU/
mL。
The present invention detects carrier and signal by the collection based on graphene oxide photo-thermal effect of above-mentioned optimization experiment preparation
Integrated thermometer (as shown in Figure 1) is resulted from, can be used for quickly detecting salmonella typhimurium.
Embodiment 4 is investigated based on the rate of recovery of thermometer detection method
After thermometer mercury head surface prepared by above-mentioned optimization modifies anti-salmonella typhimurium antibody, hurt respectively with mouse
Cold salmonella (103,105,108CFU/mL, 300 μ L) 37 DEG C be incubated for 0.5h, then rinse thermometer 3 with 0.01mol/L PBS
It is secondary, wash away unbonded bacterium.The thermometer and 37 DEG C of incubation 1h of immune graphene complex of bacterium will be captured, is rinsed with PBS
Thermometer 3 times, wash away unbonded graphene.After mercury head surface is completely dried, combined on 808nm laser irradiation thermometer
Graphene region, temperature change can recorde by thermometer.Temperature change value is brought into standard curve, it is dense to obtain this
Spending the corresponding rate of recovery is 95%~112%, meets testing requirements.
Embodiment 5 is investigated based on the selective enumeration method of thermometer detection method
After thermometer mercury head surface prepared by above-mentioned optimization modifies anti-salmonella typhimurium antibody, hurt respectively with mouse
Cold salmonella, Escherichia coli O 157: H7 and staphylococcus aureus (1 × 108CFU/mL, 300 μ L) 37 DEG C be incubated for 0.5h, use
0.01mol/L PBS is rinsed thermometer 3 times, washes away unbonded bacterium.To capture the thermometer of bacterium respectively with immune graphite
37 DEG C of incubation 1h of alkene compound are rinsed thermometer 3 times with PBS, wash away unbonded graphene.It is completely dried to mercury head surface
Afterwards, with the graphene region combined on 808nm laser irradiation thermometer, temperature change can recorde by thermometer.It can by Fig. 5
To find out, when only adding salmonella typhimurium, Escherichia coli and golden yellow are apparently higher than by the △ T that laser irradiation generates
Staphylococcus, to prove the specificity of this method preferably.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not to invention protection scope
Limitation, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not required to
It is still within the scope of the present invention to make the creative labor the various modifications or changes that can be made.
Claims (10)
1. a kind of collection detection carrier and signal based on graphene oxide photo-thermal effect results from integrated thermometer detection method,
Characterized by comprising the following steps:
(1) thermometer mercury head surface is coupled target determinand antibody
First thermometer mercury head surface is pre-processed, the thermometer mercury head handled well is inserted into antibody-solutions and is incubated
It educates, cleans；
(2) preparation of graphene is immunized
Target determinand antibody is added in graphene oxide solution and is reacted, antibody-graphene compound is obtained, i.e., it is immune
Graphene；
(3) detection of target determinand.
2. detection method according to claim 1, which is characterized in that the concrete operation method of the step (1) are as follows:
1.1 thermometer surface preparations
Detergent cleaning temperature meter mercury head surface is first used, is then cleaned with deionized water, it is dry to after anhydrous, by thermometer water
Silver-colored head is placed in ultrasound in Piranha solution, then being cleaned with a large amount of deionized water is neutral, drying to mercury head surface pH；To
Thermometer restores to room temperature, is dipped in APTES methanol solution and reacts；Then thermometer mercury head is rinsed with methanol, washed
Remaining APTES is removed, thermometer is placed in baking oven and is dried；
The coupling of 1.2 thermometer mercury head surface antibody
Target determinand antibody is added in polystyrene sample pipe, the thermometer mercury head that step (1) is handled well is inserted into
It is incubated in antibody-solutions, rinses mercury head with buffer, wash away unbonded antibody.
3. detection method according to claim 2, which is characterized in that in the step 1.1, ultrasonic time be 0.5~
1.5h, preferably 1h；Dry condition is 60~70 DEG C of dry 1.5~2.5h after the ultrasonic cleaning of thermometer mercury head, preferably
65 DEG C of dry 2h；Reaction condition are as follows: room temperature reaction 2~2.5h of time, preferably room temperature reaction time 2h；Thermometer is dried in baking oven
Dry condition is 50~60 DEG C of drying 25~35min, preferably 50 DEG C drying 30min；The volume of Piranha solution be 20~
25mL；The mass fraction of APTES methanol solution is 10%；Methanol rinse thermometer mercury head number be 2~3 times, preferably 3
It is secondary.
4. detection method according to claim 2, which is characterized in that in the step 1.2, the use of target determinand antibody
Amount is 300~350 μ L, and concentration is 3 μ g/mL；Incubation conditions are 35~40 DEG C of incubations 1~2h, preferably 37 DEG C incubation 1.5h；It is slow
Fliud flushing is 0.01mol/L PBS, is rinsed 2~3 times.
5. detection method according to claim 1, which is characterized in that reaction condition is room temperature concussion in the step (2)
React 5~7h, preferably room temperature concussion reaction 6h；The volume ratio of target determinand antibody and graphene oxide solution is 1:1, mesh
Mark determinand antibody concentration is 10 μ g/mL, and graphene oxide solution concentration is 200 μ g/mL；Obtained immune graphene is at 4 DEG C
It saves.
6. detection method according to claim 1, which is characterized in that the concrete operation method of the step (3) are as follows:
By step (1) surface modification there is target determinand antibody thermometer to be inserted into target determinand solution to be incubated for, use
Buffer rinses thermometer, washes away unbonded determinand；Then will be captured target determinand mercury head be inserted into it is immune
It is incubated in graphene solution, rinses thermometer with buffer；Laser irradiation mercury head position is used after its drying, irradiates front and back
Temperature change directly passes through temperature reading；Standard curve is established using temperature increase and target determinand number.
7. detection method according to claim 6, which is characterized in that the condition being incubated in target determinand solution
For 35~40 DEG C of incubations 1.5~2.5h, preferably 37 DEG C incubation 2h；Incubation conditions are 35~40 DEG C in immune graphene solution
It is incubated for 1.5~2.5h, preferably 37 DEG C incubation 2h；The buffer is 0.01mol/L PBS, is rinsed 3~4 times.
8. detection method according to claim 1, which is characterized in that the determinand is cancer cell, bacterium, macromolecular egg
White and small-molecule substance；Preferably, the bacterium is salmonella, and high molecular weight protein is anaphylactogen and carcinomebryonic antigen, small molecule
Substance is agricultural and veterinary chemicals residual；Preferably, the antibody can be replaced the identification materials such as molecularly imprinted polymer, aptamer；
Preferably, the thermometer can be the thermometer or temperature sensor of various models；Preferably, the graphene can be replaced it
He has the material of photo-thermal effect.
9. any detection method of claim 1~8 is in detection cancer cell, bacterium, high molecular weight protein and small-molecule substance
Application；Preferably, the bacterium is salmonella, and high molecular weight protein is anaphylactogen and carcinomebryonic antigen, and small-molecule substance is agriculture
Residue of veterinary drug.
10. a kind of coupling method of thermometer mercury head surface antibody, which comprises the following steps:
(1) thermometer surface preparation
Detergent cleaning temperature meter mercury head surface is first used, surface smut is removed, is then cleaned with deionized water, it is dry to anhydrous
Afterwards, thermometer mercury head is placed in ultrasound in Piranha solution, it is neutral for being cleaned with deionized water to mercury head surface pH, is done
It is dry；Restore after thermometer to room temperature, is dipped in APTES methanol solution and reacts；Then thermometer mercury is rinsed with methanol
Head washes away remaining APTES, thermometer is placed in baking oven and is dried；
(2) coupling of thermometer mercury head surface antibody
Target determinand antibody is added in polystyrene sample pipe, the thermometer mercury head that step (1) is handled well is inserted into
It is incubated in antibody-solutions, rinses mercury head with buffer, wash away unbonded antibody；
Preferably, in the step (1), ultrasonic time is 0.5~1.5h, preferably 1h；After the ultrasonic cleaning of thermometer mercury head
Dry condition is 60~70 DEG C of dry 1.5~2.5h, preferably 65 DEG C dry 2h；Reaction condition are as follows: the room temperature reaction time 2~
2.5h, preferably room temperature reaction time 2h；Thermometer is 50~60 DEG C of 25~35min of drying in baking oven drying condition；Preferably
50 DEG C of drying 30min；The volume of Piranha solution is 20~25mL；The mass fraction of APTES methanol solution is 10%；Methanol
The number for rinsing thermometer mercury head is 3~4 times, preferably 3 times；
Preferably, in the step (2), the dosage of target determinand antibody is 300~350 μ L, and concentration is 3 μ g/mL；It is incubated for item
Part is 35~40 DEG C of 1~2h of incubation, and preferably 37 DEG C incubation 1.5h buffers are 0.01mol/L PBS, is rinsed 2~3 times.
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