CN110068675A - A kind of Portable thermal imaging immunoassay method constructed based on photo-thermal and immuno-functionalized liposome - Google Patents
A kind of Portable thermal imaging immunoassay method constructed based on photo-thermal and immuno-functionalized liposome Download PDFInfo
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- CN110068675A CN110068675A CN201910391945.3A CN201910391945A CN110068675A CN 110068675 A CN110068675 A CN 110068675A CN 201910391945 A CN201910391945 A CN 201910391945A CN 110068675 A CN110068675 A CN 110068675A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N25/00—Investigating or analyzing materials by the use of thermal means
- G01N25/20—Investigating or analyzing materials by the use of thermal means by investigating the development of heat, i.e. calorimetry, e.g. by measuring specific heat, by measuring thermal conductivity
- G01N25/48—Investigating or analyzing materials by the use of thermal means by investigating the development of heat, i.e. calorimetry, e.g. by measuring specific heat, by measuring thermal conductivity on solution, sorption, or a chemical reaction not involving combustion or catalytic oxidation
- G01N25/4873—Investigating or analyzing materials by the use of thermal means by investigating the development of heat, i.e. calorimetry, e.g. by measuring specific heat, by measuring thermal conductivity on solution, sorption, or a chemical reaction not involving combustion or catalytic oxidation for a flowing, e.g. gas sample
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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
Abstract
Immunoassay method is imaged in the Portable thermal constructed the present invention relates to a kind of photo-thermal and immuno-functionalized liposome, belongs to sensor apparatus building and biochemical analysis technical field.The present invention is first with Ti3C2Quantum dot is as photo-thermal beacon molecule, liposome is prepared for photo-thermal functionalization liposome, in conjunction with immune molecule as container is loaded, it realizes the immuno-functionalized of photo-thermal liposome, finally realizes the specific recognition to object using immune response in the Portable thermal imaging analysis device of design.In the case where 808 nm are infrared ray excited, immunological probe introduce photo-thermal liposome can Efficient Conversion luminous energy be thermal energy so that the temperature of detection system increases.Raised temperature can be monitored by portable infrared thermal imaging camera, to realize the quantitative detection to object.This method equipment is simple, easy to carry, easy to operate, provides a kind of simple, sensitive and stable method for portable inspectiont.
Description
Technical field
Immunoassay method is imaged in the Portable thermal constructed the present invention relates to a kind of photo-thermal and immuno-functionalized liposome,
Belong to sensor apparatus building and biochemical analysis technical field.
Background technique
With the development of society, environmental monitoring, food safety and medical diagnosis on disease are increasingly valued by people, build
Many detection methods based on optics, chromatography, mass spectrum and electrochemistry are found, these detection methods have accurate, sensitive, stable
The advantages that.But equipment is expensive and the reasons such as operation is multiple limit its in terms of quick detection and daily life into one
Step application, especially in remote districts.Therefore, design a kind of equipment it is simple, it is easily operated, stablize practical detection method be tool
Significant.
Photothermal conversion has application at many aspects of life as a kind of common physical phenomenon.In During Illumination mistake
Cheng Zhong can convert light energy into thermal energy there are many material.And in these materials, Ti3C2Quantum dot is in 808 nm infrared lights
There are efficient solar-thermal conversion rate and the properties such as nontoxic under excitation.Meanwhile the quantum dot has small size and good
Good salt tolerance is a kind of material being very suitable for applied to field of bioanalysis.Many existing using temperature as sensing
Detection signal portable bio-analysis in, nanogold and ferriferrous oxide nano-particle etc. are as traditional as photo-thermal letter
Mark molecule is usually used to as the beacon molecule for generating Photothermal Signals, but poor biocompatibility and photothermal conversion efficiency be not high
The problem of limit temperature type sensing further applying in portable bio-analysis field.In recent years, by the way that beacon is divided
Son is loaded into the release and enrichment for forming a kind of material for having good biocompatibility in liposome, and realizing beacon molecule,
The utilization efficiency for improving beacon molecule, in biological medicine, there are many researchs for bio-imaging and bioanalysis etc..In order to mention
The sensitivity of high portable sensor, signal the generating resource and signal supervisory instrument are most important one two o'clocks.More than being based on
Content constructs a kind of immune bioanalysis of Portable thermal imaging for being applied to temperature type based on photo-thermal and immuno-functionalized liposome
Method must can effectively improve the sensitivity and accuracy of detection.
Summary of the invention
The purpose of the present invention is to provide the preparation methods of a kind of photo-thermal and immuno-functionalized liposome, and combine antigen anti-
Immunosensor is imaged to construct Portable thermal in body immune response.Its technical principle is by introducing Ti on immunological probe3C2Amount
The infrared light of 808 nm is changed into heat by son o'clock, and the temperature of raising can be monitored by portable infrared thermal imaging camera.When not having
When having object, photo-thermal beacon molecule is few in detection system, and temperature increase is smaller；In the presence of object, light in detection system
Hot beacon molecule increases, and temperature increase becomes larger；The variation of temperature is positively correlated in a certain range with target concentration, and can be with
The quantitative detection of rapid and handy is realized by portable infrared thermal imaging camera.
To achieve the above object, The technical solution adopted by the invention is as follows:
Immunoassay method is imaged in a kind of photo-thermal and the Portable thermal of immuno-functionalized liposome building comprising the steps of:
Step S1 Ti3C2The synthesis of quantum dot: 0.5 g Ti is taken3AlC2It is 48% that powder, which is added to mass fraction as presoma,
Hydrofluoric acid carry out 20 hours etching reactions, the precipitating after reaction is washed with deionized to neutrality.Take the above-mentioned preparation of 0.1g
Product be added in the tetramethylammonium hydroxide aqueous solution that 20 mL mass fractions are 25% and carry out 24 hours stripping reactions.It will
Reaction product is washed till neutrality, and soluble in water for the ratio of 1:150 with mass ratio.Under the protection of argon atmosphere, it will mix molten
Liquid carries out 20 hours water bath sonicators, and mixed liquor passes through the centrifuge separation supernatant of different rotating speeds after ultrasound, and by the upper of acquirement
Clear liquid saves backup in 4 DEG C.
The design of step S2 photo-thermal liposome immunization functionalization probe:
Step S21 by dipalmitoylphosphatidylcholine (DPPC), cholesterol and dipalmitoylphosphatidylethanolamine (DPPE) with
The molar ratio of 10:10:1 is mixed in chloroform (2.0-4.0 mL).After being passed through nitrogen deoxygenation and ultrasonic dissolution, mixed liquor is set
In drag, rotary evaporation is carried out until drag forms thin film at 45 DEG C.
Step S22 is subsequent, and it includes 5mg/mL Ti that 1.0-4.0 mL, which is added,3C2The phosphate buffered saline solution (10 of quantum dot
MM, pH=7.4), and quickly rotate at 45 DEG C until drag film is removed；By mixed solution with Probe Ultrasonic Searching method in ice
Ultrasound generates unilamelar liposome, ultrasonic time 10min in water-bath.
Gained liposome mixed liquor is carried out ice-water bath ultrasound, ultrasonic time 20min by step S23 again, and utilizes syringe
It is 0.44 μm of polycarbonate film to remove bulky grain liposome that liposome mixed liquor, which is pressed through aperture,.Then use
Molecular cut off is that the bag filter of 100 KDa is purified to remove the Ti not being loaded3C2Quantum dot.
Step S24 takes photo-thermal functionalization liposome (2.0-4.0mL) to be after purification slowly dropped into 3 mL mass fractions
In 2.5% glutaraldehyde water solution, incubation reaction is carried out at 25 DEG C.It is 100KDa by molecular cut off after being incubated for 1 hour
Bag filter carries out dialysis purification and removes not connected glutaraldehyde.
It includes 0.05wt% Tween-20, detection antibody that then 1.0-3.0 mL, which is added, in mixed liquor after dialysis by step S25
(PSA-173) it in phosphate buffered saline solution (10 mM, pH=7.4), and is incubated for 1 hour at 4 DEG C, takes 1.0 mL again later
Phosphate buffered saline solution (10 mM, pH=7.4) comprising 10mg/mL bovine serum albumin is closed.Last mixed liquor passes through
The liposome probe of sephadex g-100 chromatographic column progress separating-purifying, gained photo-thermal and immuno-functionalized is placed in 4 DEG C of rings
It is saved backup under border.
The building of step S3 Portable thermal imaging immunoassay apparatus: 3D printing is utilized according to the model of modeling software design
Machine prepares the portable test box of test, excitation light source and detection cell bracket.The infrared of 808 nm is installed in optical path inlet
Light source, and portable infrared thermal imaging camera is inserted into mobile phone and is used as detection system thermal signal reading device.Mobile test
The detection cell bracket of case side makes in the middle part of the intracorporal detection cell side of source alignment test box, and detection cell center location is aligned
Reading data the window's position of surface.
The building of step S4 immune response: capture antibody (20-80 μ L) is added in the high affine plate in 12 holes first and seals overnight
It closes, sequentially adds target detection thing (20-80 μ L), the functionalization liposome nano-probe (20-80 μ L) that step S2 is obtained
Hatch 30 min to form antibody-antigen-antibody interlayer type immune complex, after being washed with phosphate buffer solution, adds phosphorus
Acid buffering solution (50-100 μ L) washing.The infrared light supply for opening 808 nm is irradiated detection cell.
Step S5 output temperature signal detection: the immune molecule specific recognition object modified by lipid volume surrounding,
And probe is fixed in ELISA Plate, under 808 nanometers infrared ray excited, the Ti3C2 quantum dot that loads in liposome is by light
It can be converted into thermal energy, so that the temperature in detection system rises, and temperature is read using portable infrared thermal imaging camera and believe
Number variation come quantitative objective object concentration, finally realize Portable thermal be imaged immunoassay.
More specifically,
The Ti of preparation described in step S13C2The lateral dimension size of quantum dot is 1-5 nm, with a thickness of 1-2 nm.
The preparation process of photo-thermal liposome immunization functionalization probe described in step S2 is as shown in Figure 2.Step S2 preparation
Photo-thermal liposome immunization functionalization probe size be 190-210 nm；The quantum dot quantity of package is 400-500；Before connection
Column gland specific antibody PSA-173 quantity is 700-800.
The pictorial diagram that immunoassay apparatus is imaged in Portable thermal described in step S3 is as shown in Figure 3；Detection case internal junction
Structure is as shown in Figure 4 with size.
808 nm infrared light supply output power densities described in step S3 are 1.5W/cm-2, irradiation time is 5 minutes.
Temperature change described in step S5 is that Δ T(final temperature subtracts initial temperature and the raised temperature of solvent),
ΔT=T Max -T Surr -T H2O ；It is as shown in Figure 5 that Portable infrared imaging camera shoots photo.Thermal imaging mode is color contrast
Degree；The imaging color of measured Portable thermal imaging immunoassay apparatus increases gradually red shift with target concentration, and temperature becomes
Change numerical value to increase with target concentration and increase.
Remarkable advantage of the invention is:
Photo-thermal prepared by the present invention and immuno-functionalized liposome are sensitive to 808 nm infrared optical responses, can be by luminous energy Efficient Conversion
For thermal energy, and it is used for monitoring biological immune reaction, realizes the sensitive portable detection to Multiple Antibodies.This method equipment letter
It is single, it is easy to carry, it is easy to operate, a kind of simple, sensitive and stable method is provided for portable inspectiont.
Detailed description of the invention
Fig. 1 is a kind of Portable thermal imaging immunoassay method signal constructed based on photo-thermal and immuno-functionalized liposome
Figure；
Fig. 2 is the preparation process of photo-thermal liposome immunization functionalization probe；
Fig. 3 is that immunoassay apparatus pictorial diagram is imaged in Portable thermal；1- mobile phone, 2-FLIR one portable infrared thermal imaging are taken the photograph
As head, 3-808 nm infrared light supply, the portable test box of 4-, 5- test pond bracket；
Fig. 4 is Portable checking box perspective view;A is internal structure top view, and B is front view, and C is left view；
Fig. 5 is that immune sensing is imaged to the infrared imaging figure of temperature-responsive in Portable thermal；
Fig. 6 is by the standard working curve that does in embodiment 1 to prostate-specific antigen.
Specific embodiment
Below by specific implementation example, technical scheme is described further, but this cannot be limited with this
The range of invention.
Embodiment 1
1. Ti3C2The synthesis of quantum dot:
By 0.5 g Ti3AlC2Powder is slowly added into the hydrofluoric acid that 10 mL mass fractions are 48%, and is stirred at 60 DEG C
20 hours, gained suspension was centrifuged 10 minutes under the revolving speed of 3500 rpm, and precipitating is washed with deionized until washing molten
Liquid is in neutrality.Then, the above-mentioned washed product of 0.1 g is taken to be added to the tetramethylammonium hydroxide water that 20 mL mass fractions are 25%
It in solution, and is stirred at room temperature 24 hours, gained suspension is centrifuged 15 minutes under the revolving speed of 3500 rpm, and what is obtained is heavy
Shallow lake is washed with 100 mL deionized waters, and obtained neutral products are soluble in water with the ratio of mass ratio 1:150, and in argon atmosphere
Protection under, mixed solution is subjected to 20 hours water bath sonicators (ultrasonic power 200W).Finally, gained suspension is 5000
It is centrifuged 30 minutes under the revolving speed of rpm, gained supernatant is centrifuged 10 minutes under 12000 rpm revolving speeds again, and final supernatant is 4
It is saved backup at DEG C.
2. the design of photo-thermal and immuno-functionalized liposome probe
By dipalmitoylphosphatidylcholine (DPPC), cholesterol and dipalmitoylphosphatidylethanolamine (DPPE) with 10:10:1's
It is added in 2.0 mL chloroforms after molar ratio mixing (gross mass is 2.38 mg), and dissolves within ultrasound 10 minutes in a nitrogen atmosphere.
Mixed liquor is placed in the pear-shaped flask of 50 mL, and is depressurized (0.09 MPa) drying using rotary evaporator at 45 DEG C,
Flask inner wall is set to form thin film.Then, it includes 10 mg/mL Ti that 2.0 mL, which are added,3C2The phosphate-buffered salt of quantum dot is molten
Liquid (10 mM, pH=7.4), and quickly rotate at 45 DEG C until drag film is removed.By mixed solution Probe Ultrasonic Searching
Method carries out 10 minutes ultrasound (power 20W in ice-water bath；Pulse mode is to open for 5 seconds, is closed within 1 second).Gained mixed liquor carries out again
20 minutes water bath sonicators, and using syringe by liposome mixed liquor be pressed through aperture be 0.44 μm polycarbonate it is thin
Film is then purified using the bag filter that molecular cut off is 100 KDa.Take the photo-thermal functionalization lipid of 2.0 mL after purification
Body is slowly dropped into the glutaraldehyde water solution of 3.0 mL mass fractions 2.5%, and is incubated for 1 hour at 25 DEG C.After incubation
Mixed liquor is that 100KDa bag filter carries out dialysis 24 hours by molecular cut off.Packet then is added in the mixed liquor after dialysis
In the phosphate buffered saline solution (10 mM, pH=7.4) of the detection containing 1.0 mg/mL antibody (PSA-173), and 1 is incubated at 4 DEG C
Hour, taking 1.0 mL again later includes that the phosphate buffered saline solution (10 mM, pH=7.4) of 10mg/mL bovine serum albumin is added
Into the mixed liquor of previous step, and it is incubated for 1 hour at 4 DEG C again.Last mixed liquor is chromatographed by sephadex g-100
The liposome probe of column progress separating-purifying, gained photo-thermal and immuno-functionalized, which is placed under 4 DEG C of environment, to be saved backup.
3. immunoassay apparatus building is imaged in Portable thermal:
The portable test box of 3D printer preparation test, excitation light source and detection are utilized according to the model of modeling software design
Pond bracket.The infrared light supply of 808 nm is installed in optical path inlet, and portable infrared thermal imaging camera is inserted into mobile phone
As detection system thermal signal reading device.The detection cell bracket of mobile test case side makes source alignment test box intracorporal
In the middle part of detection cell side, and reading data the window's position right above the alignment of detection cell center location.
4. (the present embodiment carcinomebryonic antigen is analyzed as simulated target for the detection of couple object prostate-specific antigen PSA
Object)
It includes the Tween-20 and 10 μ g/mL capture that mass fraction is 0.05% that 50 μ L are added in the high affine plate in 12 holes first
The phosphate buffered saline solution of antibody (PSA-021), and closed overnight at 4 DEG C.Adding 300 μ L mass concentrations is 1.0 wt
The BSA of % is closed 1 hour.After being washed with PBS solution, sequentially adds the PSA standard sample of 50 μ L and the photo-thermal of 100 μ L and exempt from
Epidemic disease functionalized nano probe reacts 30 minutes respectively.Remove supernatant after be added 100 μ L phosphate buffered saline solutions (10 mM,
PH=7.4), and the high affine plate in 12 holes is placed on detection cell bracket, after adjusting detection cell position, open 808 nm's
Infrared light supply, irradiation time are 5 minutes, output power 1.5W/cm2.Read the temperature of portable infrared thermal imaging camera
Variation is to quantify.The resulting temperature change of its result is to the standard working curve of PSA as shown in fig. 6, the range of linearity is 1.0-50
Ng/mL, detection are limited to 0.4 ng/mL.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (9)
1. immunoassay method is imaged in a kind of Portable thermal constructed based on photo-thermal and immuno-functionalized liposome, feature exists
In: described method includes following steps:
Step S1 Ti3C2The synthesis of quantum dot；
The design of step S2 photo-thermal liposome immunization functionalization probe；
The building of step S3 Portable thermal imaging immunoassay apparatus；
The building of step S4 immune response: antibody-antigene-nano-probe immuno-sandwich is formed in high affine 12 hole elisa Plates
Structure；
Step S5 output temperature signal detection.
2. a kind of Portable thermal imaging constructed based on photo-thermal and immuno-functionalized liposome according to claim 1 is immune
Analysis method: Ti described in step S13C2Quantum dot is synthesized by hf etching Ti3AlC2Presoma, and use tetramethyl
Base ammonium hydroxide remove；The Ti of preparation3C2Lateral size of dots is 1-5 nm, with a thickness of 1-2 nm.
3. a kind of Portable thermal imaging constructed based on photo-thermal and immuno-functionalized liposome according to claim 1 is immune
Analysis method, it is characterised in that: photo-thermal liposome immunization functionalization probe described in step S2 is will by film dispersion method
Ti3C2Quantum dot is loaded into liposome, and is connected to prostate specific antibody PSA-173 by Euplotes woodruffi
Photo-thermal surface of liposome is made.
4. a kind of Portable thermal imaging constructed based on photo-thermal and immuno-functionalized liposome according to claim 1 is immune
Analysis method, it is characterised in that: the photo-thermal liposome immunization functionalization probe size of step S2 preparation is 190-210 nm；Package
Quantum dot quantity be 400-500；The prostate specific antibody PSA-173 quantity of connection is 700-800.
5. a kind of Portable thermal imaging constructed based on photo-thermal and immuno-functionalized liposome according to claim 1 is immune
Analysis method, it is characterised in that: immunoassay apparatus is imaged by the infrared light supply, just of 808 nm in Portable thermal described in step S3
Formula test box, detection cell and portable infrared thermal imaging camera FLIR one is taken to assemble；Portable test box is by 3D
Printer is made.
6. a kind of Portable thermal imaging constructed based on photo-thermal and immuno-functionalized liposome according to claim 1 is immune
Analysis method, it is characterised in that: ELISA Plate is the detection cell in analytical equipment in step S4；Detection identification process, which relies on, to be formed
Antibody-antigene-nano-probe interlayer type immune complex is realized.
7. immunoassay is imaged in the Portable thermal according to claim 1 constructed based on photo-thermal and immuno-functionalized liposome
Method, it is characterised in that: step S4 process specifically: capture antibody 20-80 μ L is added in the high affine plate in 12 holes and seals overnight
It closes, sequentially adds target detection thing 20-80 μ L, nano-probe 20-80 μ L exempts to form antibody-antigen-antibody interlayer type
Epidemic disease compound hatches 30 min.
8. a kind of Portable thermal imaging constructed based on photo-thermal and immuno-functionalized liposome according to claim 1 is immune
Analysis method, it is characterised in that: step S5 specifically: the immune molecule specific recognition target modified by lipid volume surrounding
Object, and probe is fixed in ELISA Plate, under 808 nanometers infrared ray excited, the Ti that is loaded in liposome3C2Quantum dot will
Luminous energy is converted into thermal energy, so that the temperature in detection system rises, and reads temperature using portable infrared thermal imaging camera
Signal intensity carrys out quantitative objective object concentration, finally realizes that immunoassay is imaged in Portable thermal.
9. a kind of Portable thermal imaging constructed based on photo-thermal and immuno-functionalized liposome according to claim 8 is immune
Analysis method, it is characterised in that: 808 nanometers of the infrared optical output power is 1.5 W/cm-2, light application time 300
s。
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| CN113552199B (en) * | 2021-07-29 | 2023-06-20 | 四川农业大学 | FeS-based 2 Molecularly imprinted electrochemical sensor with/C/MQDs/GCE modified electrode and preparation method thereof |
| CN114455690A (en) * | 2021-12-23 | 2022-05-10 | 南京师范大学 | Magnetic Mxene microbial carrier material and preparation method thereof |
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