CN110068675B - Portable thermal imaging immunoassay method based on photo-thermal and immune functionalized liposome construction - Google Patents

Portable thermal imaging immunoassay method based on photo-thermal and immune functionalized liposome construction Download PDF

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CN110068675B
CN110068675B CN201910391945.3A CN201910391945A CN110068675B CN 110068675 B CN110068675 B CN 110068675B CN 201910391945 A CN201910391945 A CN 201910391945A CN 110068675 B CN110068675 B CN 110068675B
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唐点平
蔡国能
余镇重
张莉佳
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Fuzhou University
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Abstract

The invention relates to a portable thermal system constructed by photo-thermal and immune functional liposomeAn image immunoassay method belongs to the technical field of sensor instrument construction and biochemical analysis. The invention firstly uses Ti3C2The quantum dots are used as photothermal beacon molecules, the liposome is used as a loading container, photothermal functionalized liposome is prepared, immune molecules are combined to realize the immune functionalization of the photothermal liposome, and finally, the immune reaction is utilized in a designed portable thermal imaging analysis device to realize the specific recognition of a target object. Under the excitation of 808 nm infrared light, the photothermal liposome introduced by the immune probe can efficiently convert light energy into heat energy, so that the temperature of the detection system is increased. The increased temperature can be monitored by a portable infrared thermal imaging camera, so that quantitative detection of the target object is realized. The method has the advantages of simple equipment, portability and simple operation, and provides a simple, sensitive and stable method for portable detection.

Description

Portable thermal imaging immunoassay method based on photo-thermal and immune functionalized liposome construction
Technical Field
The invention relates to a portable thermal imaging immunoassay method for constructing a photothermal and immune functionalized liposome, belonging to the technical field of construction of a sensing instrument and biochemical analysis.
Background
With the development of society, environmental monitoring, food safety and disease diagnosis are more and more emphasized by people, and a plurality of detection methods based on optics, chromatography, mass spectrometry and electrochemistry are established, and have the advantages of accuracy, sensitivity, stability and the like. However, the expensive equipment and the complex operation limit the further application of the equipment in the aspects of rapid detection, daily life detection and the like, especially in remote areas. Therefore, it is important to design a stable and practical detection method with simple equipment and easy operation.
Photothermal conversion is a common physical phenomenon, and has applications in many aspects of life. During the illumination process, there are many materialsThe material can convert light energy into heat energy. And among these, Ti3C2The quantum dots have high photo-thermal conversion rate, no toxicity, no harm and other properties under the excitation of 808 nm infrared light. Meanwhile, the quantum dot has tiny size and good salt resistance, and is a material very suitable for being applied to the field of biological analysis. In many existing portable bioanalysis taking temperature as a sensing signal, nanogold, ferroferric oxide nanoparticles and the like serving as traditional photothermal beacon molecules are generally used as the beacon molecules for generating photothermal signals, but the further application of temperature type sensing in the field of portable bioanalysis is limited due to the problems of poor biocompatibility and low photothermal conversion efficiency. In recent years, a material with good biocompatibility is formed by loading beacon molecules into liposomes, the release and enrichment of the beacon molecules are realized, the utilization efficiency of the beacon molecules is improved, and a lot of researches are carried out on the aspects of biomedicine, biological imaging, biological analysis and the like. In order to improve the sensitivity of the portable sensor, the signal generation source and the signal detection device are two of the most important. Based on the above contents, the method for constructing the portable thermal imaging immunobiological analysis based on the photothermal and immune functionalized liposome applied to the temperature type can effectively improve the sensitivity and the accuracy of detection.
Disclosure of Invention
The invention aims to provide a preparation method of a photothermal and immune functionalized liposome, and a portable thermal imaging immunosensor is constructed by combining antigen-antibody immunoreaction. The technical principle is that Ti is introduced into an immune probe3C2The quantum dots convert 808 nm infrared light into heat, and the increased temperature can be monitored by the portable infrared thermal imaging camera. When no target object exists, the number of photo-thermal beacon molecules in the detection system is small, and the temperature increase is small; when the target exists, the number of photo-thermal beacon molecules in the detection system is increased, and the temperature increase is increased greatly; the temperature change and the concentration of the target object form positive correlation in a certain range, and the portable infrared thermal imaging camera can be used for realizing quick and portable quantitative detection.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a portable thermal imaging immunoassay method constructed by photothermal and immune functionalized liposome comprises the following steps:
step S1 Ti3C2And (3) synthesis of quantum dots: take 0.5 g Ti3AlC2And adding the powder serving as a precursor into hydrofluoric acid with the mass fraction of 48% for etching reaction for 20 hours, and washing the reacted precipitate to be neutral by using deionized water. 0.1g of the product prepared above was added to 20 mL of an aqueous 25% tetramethylammonium hydroxide solution to conduct a stripping reaction for 24 hours. The reaction product was washed to neutrality and dissolved in water in a mass ratio of 1: 150. And (3) carrying out water bath ultrasound on the mixed solution for 20 hours under the protection of argon atmosphere, centrifuging the mixed solution at different rotating speeds after the ultrasound treatment to separate supernatant, and storing the obtained supernatant at 4 ℃ for later use.
Step S2 design of photothermal liposome immune functional probe:
step S21 Dipalmitoylphosphatidylcholine (DPPC), cholesterol, and Dipalmitoylphosphatidylethanolamine (DPPE) were mixed in chloroform (2.0-4.0 mL) at a molar ratio of 10:10: 1. After nitrogen is introduced to remove oxygen and ultrasonic dissolution is carried out, the mixed solution is placed at the bottom of a flask, and rotary evaporation is carried out at 45 ℃ until a thin film is formed at the bottom of the flask.
Step S22 subsequently, 1.0-4.0 mL of Ti-containing solution containing 5mg/mL of Ti was added3C2Phosphate buffered saline (10 mM, pH =7.4) of quantum dots and rapidly rotated at 45 ℃ until the bottom film of the flask peeled off; and ultrasonically treating the mixed solution in an ice-water bath by using a probe ultrasonic method to generate the unilocular liposome, wherein the ultrasonic time is 10 min.
And step S23, performing ice-water bath ultrasound on the obtained liposome mixed solution for 20min, and extruding the liposome mixed solution through a polycarbonate film with the aperture of 0.44 mu m by using a syringe to remove large-particle liposomes. Subsequent purification using dialysis bags with a molecular weight cut-off of 100kDa to remove unloaded Ti3C2And (4) quantum dots.
Step S24, the purified photo-thermal functionalized liposome (2.0-4.0 mL) is slowly dropped into 3 mL of glutaraldehyde aqueous solution with the mass fraction of 2.5%, and incubation reaction is carried out at 25 ℃. After 1 hour of incubation, the unattached glutaraldehyde was removed by dialysis purification through a dialysis bag with a molecular weight cut-off of 100 kDa.
Step S25 the dialyzed mixture was then added to 1.0-3.0 mL of phosphate buffered saline (10 mM, pH =7.4) containing 0.05wt% tween-20, detection antibody (PSA-173) and incubated at 4 ℃ for 1 hour, after which 1.0 mL of phosphate buffered saline (10 mM, pH =7.4) containing 10mg/mL bovine serum albumin was blocked. And finally, separating and purifying the mixed solution by a sephadex G-100 chromatographic column, and storing the obtained photothermal and immune functionalized liposome probe in an environment at 4 ℃ for later use.
Step S3 construction of the portable thermal imaging immunoassay device: and preparing a portable test box for testing, an excitation light source and a detection pool support by using a 3D printer according to a model designed by modeling software. An infrared light source with 808 nm is arranged at an entrance of a light path, and a portable infrared thermal imaging camera is inserted into a mobile phone to be used as a thermal signal reading device of the detection system. And the detection pool support on the side surface of the test box is moved to enable the light source to be aligned to the middle of the side surface of the detection pool in the test box, and the circle center position of the detection pool is aligned to the position of the data reading window right above the detection pool.
Step S4 construction of immune response: first, capture antibody (20-80 μ L) is added into a 12-hole high affinity plate for overnight sealing, then target detection object (20-80 μ L) and functionalized liposome nanoprobe (20-80 μ L) obtained in step S2 are sequentially added to form antibody-antigen-antibody sandwich type immune complex for incubation for 30 min, and after washing with phosphate buffer solution, phosphate buffer solution (50-100 μ L) is added for washing. And turning on an infrared light source with 808 nm to irradiate the detection cell.
Step S5 outputs temperature signal detection: the target object is specifically identified through immune molecules modified on the outer layer of the liposome, the probe is fixed in an enzyme label plate, under the excitation of infrared light of 808 nanometers, the Ti3C2 quantum dots loaded in the liposome convert light energy into heat energy, so that the temperature in a detection system rises, a portable infrared thermal imaging camera is used for reading the change of temperature signals to quantify the concentration of the target object, and finally, the portable thermal imaging immunoassay is realized.
More specifically, the present invention is to provide a novel,
ti prepared as described in step S13C2The quantum dots have a transverse dimension of 1-5 nm and a thickness of 1-2 nm.
The preparation process of the photothermal liposome immune functionalized probe described in step S2 is shown in FIG. 2. The size of the photothermal liposome immune functional probe prepared in the step S2 is 190-210 nm; the number of the wrapped quantum dots is 400-500; the amount of the linked prostate specific antibody PSA-173 was 700-800.
A physical diagram of the portable thermal imaging immunoassay device described in step S3 is shown in fig. 3; the internal structure and dimensions of the detection box are shown in fig. 4.
The output power density of the 808 nm infrared light source in the step S3 is 1.5W/cm-2The irradiation time was 5 minutes.
The temperature change described in step S5 is Δ T (final temperature minus initial temperature and temperature of solvent rise), ΔT=T Max -T Surr -T H2O (ii) a The portable infrared imaging camera takes a picture as shown in fig. 5. The thermal imaging mode is color contrast; the measured imaging color of the portable thermal imaging immunoassay device gradually red shifts along with the increase of the concentration of the target object, and the temperature change value increases along with the increase of the concentration of the target object.
The invention has the following remarkable advantages:
the photothermal and immune functionalized liposome prepared by the invention is sensitive to 808 nm infrared light response, can efficiently convert light energy into heat energy, and is used for monitoring biological immune reaction, thereby realizing sensitive and portable detection of various antibodies. The method has the advantages of simple equipment, portability and simple operation, and provides a simple, sensitive and stable method for portable detection.
Drawings
FIG. 1 is a schematic diagram of a portable thermal imaging immunoassay method based on photothermal and immune functionalized liposome construction;
FIG. 2 is a process for preparing a photothermal liposome immune functionalized probe;
FIG. 3 is a pictorial view of a portable thermographic immunoassay device; 1-a mobile phone, 2-a FLIR one portable infrared thermal imaging camera, a 3-808 nm infrared light source, 4-a portable test box and 5-a test pool support;
FIG. 4 is a perspective view of the portable inspection box, wherein A is a top view of the internal structure, B is a front view, and C is a left view;
FIG. 5 is a graph of infrared imaging of the response of a portable thermographic immunosensor to temperature;
FIG. 6 is a standard working curve for prostate specific antigen as in example 1.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific implementation examples, but the scope of the present invention is not limited thereby.
Example 1
1. Ti3C2And (3) synthesis of quantum dots:
0.5 g of Ti3AlC2The powder was slowly added to 10 mL of 48% by mass hydrofluoric acid and stirred at 60 ℃ for 20 hours, the resulting suspension was centrifuged at 3500 rpm for 10 minutes, and the precipitate was washed with deionized water until the wash solution was neutral. Subsequently, 0.1g of the above washing product was added to 20 mL of an aqueous solution of tetramethylammonium hydroxide with a mass fraction of 25%, and stirred at room temperature for 24 hours, the obtained suspension was centrifuged at 3500 rpm for 15 minutes, the obtained precipitate was washed with 100 mL of deionized water, the obtained neutral product was dissolved in water at a mass ratio of 1:150, and the mixed solution was subjected to water bath ultrasound (ultrasonic power of 200W) for 20 hours under the protection of argon atmosphere. Finally, the suspension was centrifuged at 5000 rpm for 30 minutes, the supernatant was centrifuged at 12000 rpm for 10 minutes and the final supernatant was stored at 4 ℃ until use.
2. Design of photothermal and immune functionalized liposome probe
Mixing Dipalmitoylphosphatidylcholine (DPPC), cholesterol and Dipalmitoylphosphatidylethanolamine (DPPE) at a molar ratio of 10:10:1After mixing (total mass: 2.38 mg), the mixture was added to 2.0 mL of chloroform and dissolved by sonication for 10 minutes under a nitrogen atmosphere. The mixture was placed in a 50 mL pear-shaped flask and dried at 45 ℃ under reduced pressure (0.09 MPa) using a rotary evaporator to form a thin film on the inner wall of the flask. Subsequently, 2.0 mL of a solution containing 10mg/mL Ti was added3C2Phosphate buffered saline (10 mM, pH =7.4) of quantum dots and spun rapidly at 45 ℃ until the bottom film of the flask peeled off. The mixed solution was subjected to ultrasonic treatment in an ice-water bath for 10 minutes by a probe ultrasonic method (power 20W; pulse mode 5 sec on and 1 sec off). The resulting mixture was subjected to water bath ultrasound for another 20 minutes, and the liposome mixture was extruded through a polycarbonate film having a pore size of 0.44 μm using a syringe, followed by purification using a dialysis bag having a molecular weight cutoff of 100 KDa. 2.0 mL of the purified photothermal functionalized liposome was slowly dropped into 3.0 mL of a glutaraldehyde aqueous solution with a mass fraction of 2.5%, and incubated at 25 ℃ for 1 hour. The incubated mixture was dialyzed for 24 hours in dialysis bags with a molecular weight cut-off of 100 kDa. The dialyzed mixture was then added to a phosphate buffered saline solution (10 mM, pH =7.4) containing 1.0 mg/mL detection antibody (PSA-173) and incubated at 4 ℃ for 1 hour, after which 1.0 mL of a phosphate buffered saline solution (10 mM, pH =7.4) containing 10mg/mL bovine serum albumin was added to the mixture of the previous step and further incubated at 4 ℃ for 1 hour. And finally, separating and purifying the mixed solution by a sephadex G-100 chromatographic column, and storing the obtained photothermal and immune functionalized liposome probe in an environment at 4 ℃ for later use.
3. The construction of the portable thermal imaging immunoassay device comprises the following steps:
and preparing a portable test box for testing, an excitation light source and a detection pool support by using a 3D printer according to a model designed by modeling software. An infrared light source with 808 nm is arranged at an entrance of a light path, and a portable infrared thermal imaging camera is inserted into a mobile phone to be used as a thermal signal reading device of the detection system. And the detection pool support on the side surface of the test box is moved to enable the light source to be aligned to the middle of the side surface of the detection pool in the test box, and the circle center position of the detection pool is aligned to the position of the data reading window right above the detection pool.
4. Detection of target prostate specific antigen PSA (carcinoembryonic antigen as model target analyte in this example)
First 50 μ L of phosphate buffered saline containing 0.05% by mass of Tween-20 and 10 μ g/mL of capture antibody (PSA-021) was added to a 12-well high affinity plate and blocked overnight at 4 ℃. Then adding 300 muL of BSA with the mass concentration of 1.0 wt% for blocking for 1 hour. After washing with PBS solution, 50 muL of PSA standard sample and 100 muL of photothermal and immune functionalized nanoprobe are sequentially added, and reaction is carried out for 30 minutes respectively. After removing the supernatant, 100 μ L phosphate buffered saline (10 mM, pH =7.4) was added, and a 12-well high affinity plate was placed on the detection cell holder, after the detection cell position was adjusted, an infrared light source of 808 nm was turned on, the irradiation time was 5 minutes, and the output power was 1.5W/cm2. And reading the temperature change of the portable infrared thermal imaging camera to quantify. The standard operating curve of the PSA with respect to temperature change obtained as a result is shown in FIG. 6, and the linear range is 1.0-50 ng/mL, and the detection limit is 0.4 ng/mL.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (2)

1. A portable thermal imaging immunoassay method based on photothermal and immune functionalized liposome construction is characterized in that: the method comprises the following steps:
step S1 Ti3C2Synthesizing quantum dots;
step S2, designing a photothermal liposome immune functional probe;
step S3, constructing a portable thermal imaging immunoassay device;
step S4 construction of immune response: forming an antibody-antigen-nano probe immune sandwich structure on a high-affinity 12-hole enzyme label plate;
step S5, outputting temperature signal detection;
ti described in step S13C2The synthesis of quantum dots is carried out by etching Ti with hydrofluoric acid3AlC2The precursor is stripped by tetramethyl ammonium hydroxideForming; prepared Ti3C2The diameter of the quantum dot is 1-5 nm, and the thickness of the quantum dot is 1-2 nm;
the photothermal liposome immune functional probe of step S2 is prepared by dispersing Ti by thin film dispersion method3C2The quantum dots are loaded into the liposome, and the prostate specific antibody PSA-173 is connected to the surface of the photothermal liposome by a glutaraldehyde crosslinking method, and the method specifically comprises the following steps:
step S21, mixing dipalmitoylphosphatidylcholine DPPC, cholesterol and dipalmitoylphosphatidylethanolamine DPPE in a molar ratio of 10:10:1 in chloroform 2.0-4.0 mL; after nitrogen is introduced to remove oxygen and ultrasonic dissolution is carried out, the mixed solution is placed at the bottom of a flask, and rotary evaporation is carried out at 45 ℃ until a layer of film is formed at the bottom of the flask;
step S22 subsequently, 1.0-4.0 mL of Ti-containing solution containing 5mg/mL of Ti was added3C210 mM phosphate buffered saline solution of quantum dots, pH =7.4, and rapidly rotated at 45 ℃ until the bottom film of the flask is peeled off; ultrasonically treating the mixed solution in an ice-water bath by using a probe ultrasonic method to generate a single-chamber liposome, wherein the ultrasonic time is 10 min;
step S23, performing ice-water bath ultrasound on the obtained liposome mixed solution for 20min, and extruding the liposome mixed solution through a polycarbonate film with the aperture of 0.44 mu m by using an injector to remove large-particle liposomes; subsequent purification using dialysis bags with a molecular weight cut-off of 100kDa to remove unloaded Ti3C2Quantum dots;
s24, slowly dripping 2.0-4.0 mL of the purified photo-thermal functionalized liposome into 3 mL of glutaraldehyde aqueous solution with the mass fraction of 2.5%, and carrying out incubation reaction at 25 ℃; after 1 hour of incubation, the unattached glutaraldehyde was removed by dialysis purification through a dialysis bag with a molecular weight cut-off of 100 KDa;
step S25 the dialyzed mixture was then added to 1.0-3.0 mL of 10 mM, pH =7.4 phosphate buffered saline containing 0.05wt% tween-20, detection antibody PSA-173 and incubated at 4 ℃ for 1 hour, after which 1.0 mL of 10 mM, pH =7.4 phosphate buffered saline containing 10mg/mL bovine serum albumin was taken for blocking; finally, separating and purifying the mixed solution through a sephadex G-100 chromatographic column, and storing the obtained photothermal and immune functionalized liposome probe in an environment at 4 ℃ for later use;
the size of the photothermal liposome immune functional probe prepared in the step S2 is 190-210 nm; the number of the wrapped quantum dots is 400-500; the number of the connected prostate specific antibody PSA-173 is 700-800;
the portable thermal imaging immunoassay device in the step S3 is assembled by an infrared light source of 808 nm, a portable test box, a detection cell and a portable infrared thermal imaging camera FLIR one; the portable test box is made of a 3D printer;
the ELISA plate in the step S4 is a detection pool in the analysis device; the detection and recognition process is realized by forming an antibody-antigen-nano probe sandwich type immune complex;
the step S4 specifically includes: adding 20-80 μ L of capture antibody into a 12-hole high affinity plate, sealing overnight, and sequentially adding 20-80 μ L of target detection object and 20-80 μ L of nano probe to form antibody-antigen-antibody sandwich type immune complex for incubation for 30 min;
step S5 specifically includes: target object is specifically identified through immune molecules modified by liposome outer layer, a probe is fixed in an enzyme label plate, and Ti loaded in liposome is excited by infrared light of 808 nanometers3C2The quantum dots convert light energy into heat energy, so that the temperature in the detection system rises, the portable infrared thermal imaging camera is used for reading the temperature signal change to quantify the concentration of the target object, and finally, the portable thermal imaging immunoassay is realized.
2. The method of claim 1, wherein the method comprises the steps of: the output power of the 808 nm infrared light is 1.5W/cm-2The illumination time was 300 s.
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