CN109060178B - Thermometer rapid detection method integrating detection carrier and signal generation - Google Patents
Thermometer rapid detection method integrating detection carrier and signal generation Download PDFInfo
- Publication number
- CN109060178B CN109060178B CN201810725536.8A CN201810725536A CN109060178B CN 109060178 B CN109060178 B CN 109060178B CN 201810725536 A CN201810725536 A CN 201810725536A CN 109060178 B CN109060178 B CN 109060178B
- Authority
- CN
- China
- Prior art keywords
- thermometer
- antibody
- solution
- mercury head
- graphene oxide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
- G01K13/00—Thermometers specially adapted for specific purposes
Landscapes
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The invention discloses a thermometer detection method integrating detection carrier and signal generation based on graphene oxide photothermal effect, which is a method for sensitively and rapidly detecting a target object to be detected by using a thermometer as a carrier and establishing based on the characteristics of a nano material. The thermometer is used as a carrier of the detection method, and can read the temperature change generated by the photothermal effect, thereby simplifying the operation steps of the whole method, greatly reducing the detection cost, and being obviously superior to the immune test strip in detection sensitivity and beneficial to the wide application of the detection method.
Description
Technical Field
The invention relates to a thermometer rapid detection method integrating detection carrier and signal generation, belonging to the technical field of detection.
Background
The establishment of a simple, convenient and sensitive method for rapidly detecting food hazards is an effective way for guaranteeing food safety. The existing established rapid detection method for the hazardous substances in the food mostly uses an enzyme-linked immunosorbent assay, an immune test strip, an immunosensor and the like as carriers. The enzyme-linked immunoassay has relatively high sensitivity but complex operation, and needs an enzyme-linked immunosorbent assay instrument which is not suitable for quick detection on site. The test strip method is simple and convenient to operate and low in cost, qualitative or semi-quantitative analysis can be obtained by adopting simple detection equipment such as visual measurement or a card reader, but the detection method is relatively low in sensitivity. In addition, the composition of a carrier nitrocellulose membrane commonly used by the test strip is complex, the combination of the membrane and proteins such as antibodies and the like is influenced by various factors, and the sensitivity and the stability of the test strip are required to be improved due to the reasons of fragility, easiness in folding, scratch generation and the like in the operation process. Therefore, the search for stable and suitable detection carriers becomes a research hotspot of researchers.
The photothermal effect is mainly applied to the aspects of photothermal therapy, biological imaging and the like of cancers at present, and the research for directly applying the photothermal effect to the establishment of a detection method is less. Dovichi et al use the thermal lens effect generated by laser irradiation for thermal tracking analysis, and are the earliest studies to apply the photothermal effect to the establishment of the detection method. Qin et al establish a standard curve of the temperature rise value and the concentration of the substance to be detected by using the photothermal effect of colloidal gold, and compared with a test strip visual detection method, the method has the advantage that the detection sensitivity is improved by 32 times. Huang et al applied photothermal effect to detect 2,4, 6-trinitrotoluene with a detection limit of 14 ng/cm. In the study work before this group of subjects: zhou et al established a detection method of a cancer cell percolation test paper strip based on the photothermal effect of a nano composite material, and the detection limit can reach 600 cells. Zhang et al established a circulating tumor cell detection method based on graphene oxide functionalized immunomagnetic beads, and established a standard curve between the temperature rise value after graphene oxide irradiation and the number of circulating tumor cells, and the detection limit can reach 100 cells. However, in the above related detection method established by using the photo-thermal effect, the temperature change needs to be recorded by a thermal imager or read by an infrared temperature measuring gun, which increases the experiment cost.
Photothermal conversion materials have been widely used for diagnosis and photothermal therapy of tumors. However, in the current detection method, relatively few researches are carried out to directly apply the photothermal effect of the photothermal material to establish a rapid detection method, and the cost of an instrument (thermal imager) for detecting the temperature in the related detection method established by the photothermal effect is high (about 4 ten thousand yuan), so that the wide usability and the popularity of the instrument are limited. Therefore, it is urgent to develop a rapid detection method which is low in cost, simple in operation, and capable of rapidly integrating the carrier function and the detection function.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention aims to provide a method for rapidly detecting a thermometer based on graphene oxide photothermal effect. The invention relates to a sensitive and rapid target object detection method which is established based on the photothermal effect of a nano material by using a thermometer as a carrier. The thermometer (about 60 yuan) is used as an experiment carrier, and can read the temperature change generated by the photothermal effect, thereby simplifying the operation steps, greatly reducing the detection cost and being beneficial to the wide application of the detection method.
The second purpose of the invention is to provide a coupling method of the antibody on the surface of the mercury head of the thermometer. The target substance antibody is modified on the surface of the mercury head of the thermometer, so that the target substance antibody can be specifically captured, and a foundation is laid for improving the sensitivity of the detection method.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides a coupling method of a thermometer mercury head surface antibody integrating detection carrier and signal generation, which comprises the following steps:
(1) thermometer surface pretreatment
Cleaning the surface of the mercury head of the thermometer by using a cleaning agent to remove dirt on the surface, then cleaning by using deionized water, drying until no water exists, putting the mercury head of the thermometer into Piranha solution for ultrasonic treatment, cleaning by using a large amount of deionized water until the pH value on the surface of the mercury head is neutral, and drying; after the thermometer is returned to room temperature, the thermometer is immersed into a 3-aminopropyltriethoxysilane (3-aminopropyl) triethoxysilane (APTES) methanol solution for reaction; then washing the mercury head of the thermometer by using methanol, washing away residual APTES, and placing the thermometer in an oven for drying;
(2) coupling of thermometer mercury head surface antibody
Adding the target substance antibody to be detected into a polystyrene sample tube, inserting the thermometer silver head treated in the step (1) into an antibody solution for incubation, washing the silver head with a buffer solution, and washing away the unbound antibody.
Preferably, in the step (1), the ultrasonic time is 0.5-1.5 h; further preferably 1 hour.
Preferably, in the step (1), the drying condition of the mercury head of the thermometer after ultrasonic cleaning is 60-70 ℃ for 1.5-2.5 h, and preferably 65 ℃ for 2 h.
Preferably, in the step (1), the reaction conditions are as follows: reacting at room temperature for 2-2.5 h; further preferably, the reaction time is 2 hours at room temperature.
Preferably, in the step (1), the thermometer is dried for 25-35 min at the drying condition of 50-60 ℃ in an oven; further preferably dried at 50 ℃ for 30 min.
Preferably, in the step (1), the volume of the Piranha solution is 20-25 mL.
Preferably, in the step (1), the mass fraction of the APTES methanol solution is 10%.
Preferably, in the step (1), the number of times of washing the mercury head of the thermometer with methanol is 2-3 times, and more preferably 3 times.
Preferably, in the step (2), the dosage of the antibody solution is 300-350 μ L, and the concentration is 3 μ g/mL.
Preferably, in the step (2), the incubation condition is incubation for 1-2 h at 35-40 ℃; further preferably, the incubation is carried out at 37 ℃ for 1.5 hours.
Preferably, in the step 1.2, the buffer solution is 0.01mol/L PBS, and the washing is performed for 2-3 times.
The invention provides a graphene oxide photothermal effect-based thermometer detection method integrating detection carrier and signal generation, which comprises the following steps:
(1) thermometer mercury head surface coupling target analyte antibody
After the surface of the mercury head of the thermometer is pretreated, the treated mercury head of the thermometer is inserted into an antibody solution for incubation and cleaning;
(2) preparation of immune graphene
Adding a target substance antibody to be detected into a graphene oxide solution for reaction to obtain an antibody-graphene compound, namely immune graphene;
(3) and (5) detecting a target object to be detected.
The specific operation method of the step (1) comprises the following steps:
1.1 thermometer surface pretreatment
Cleaning the surface of the mercury head of the thermometer by using a cleaning agent to remove dirt on the surface, then cleaning by using deionized water, drying until no water exists, putting the mercury head of the thermometer into Piranha solution for ultrasonic treatment, cleaning by using a large amount of deionized water until the pH value on the surface of the mercury head is neutral, and drying; after the thermometer is returned to room temperature, the thermometer is immersed into a 3-aminopropyltriethoxysilane (3-aminopropyl) triethoxysilane (APTES) methanol solution for reaction; then washing the mercury head of the thermometer by using methanol, washing away residual APTES, and placing the thermometer in an oven for drying;
1.2 coupling of Mercury head surface antibodies to thermometer
Adding the target substance antibody to be detected into a polystyrene sample tube, inserting the thermometer silver head treated in the step (1) into an antibody solution for incubation, washing the silver head with a buffer solution, and washing away the unbound antibody.
Preferably, in the step 1.1, the ultrasonic time is 0.5-1.5 h; further preferably 1 hour.
Preferably, in the step 1.1, the drying condition of the mercury head of the thermometer after ultrasonic cleaning is 60-70 ℃ for 1.5-2.5 h, and preferably 65 ℃ for 2 h.
Preferably, in the step 1.1, the reaction conditions are as follows: reacting at room temperature for 2-2.5 h; further preferably, the reaction time is 2 hours at room temperature.
Preferably, in the step 1.1, the thermometer is dried for 25-35 min at the drying condition of 50-60 ℃ in an oven; further preferably dried at 50 ℃ for 30 min.
Preferably, in the step 1.1, the volume of the Piranha solution is 20-25 mL.
Preferably, in the step 1.1, the mass fraction of the APTES methanol solution is 10%.
Preferably, in the step 1.1, the number of times of washing the mercury head of the thermometer with methanol is 2-3 times, and more preferably 3 times.
Preferably, in the step 1.2, the dosage of the target analyte antibody is 300-350 μ L, and the concentration is 3 μ g/mL.
Preferably, in the step 1.2, the incubation condition is incubation for 1-2 hours at 35-40 ℃; further preferably, the incubation is carried out at 37 ℃ for 1.5 hours.
Preferably, in the step 1.2, the buffer solution is 0.01mol/L PBS, and the washing is performed for 2-3 times.
And (3) in the step (2), storing the obtained immune graphene at 4 ℃.
Preferably, the reaction condition is room temperature oscillation reaction for 5-7 h; further preferably, the reaction is carried out at room temperature with shaking for 6 hours.
Preferably, the volume ratio of the target analyte bacterium antibody to the graphene oxide solution is 1: 1, the concentration of the target to-be-detected bacterium antibody is 10 mug/mL, and the concentration of the graphene oxide solution is 200 mug/mL.
The specific operation method of the step (3) comprises the following steps:
inserting the thermometer with the surface modified with the target to-be-detected object antibody in the step (1) into a target to-be-detected object solution for incubation, washing the mercury head of the thermometer with a buffer solution, and washing away the unbound to-be-detected object; then inserting the mercury head capturing the target substance to be detected into an immune graphene solution for incubation, and washing the thermometer with a buffer solution; after the mercury is dried, irradiating the position of the mercury head by laser, and directly reading the temperature change before and after irradiation by a thermometer; and establishing a standard curve by using the temperature rise value and the concentration of the target object to be measured.
Preferably, the incubation condition of the target substance to be detected is 35-40 ℃ for 1.5-2.5 h; further preferably, the incubation is carried out at 37 ℃ for 2 hours.
Preferably, the incubation condition in the immune graphene solution is incubation for 1.5-2.5 h at 35-40 ℃; further preferably incubated at 37 ℃ for 2h,
preferably, the buffer solution is 0.01mol/L PBS, and the washing is carried out for 3-4 times.
The substance to be detected is cancer cell, bacteria, macromolecular protein and micromolecular substance.
Preferably, the bacteria are salmonella, the macromolecular proteins are allergens and carcinoembryonic antigens, and the micromolecular substances are pesticide residue;
preferably, the invention also falls within the protection scope of the invention, in which graphene can be replaced by photo-thermal conversion materials such as colloidal gold, nano-magnetic spheres and the like.
Preferably, the thermometer holder used in the present invention may be various types of thermometers.
Preferably, the recognition material in the present invention may be an antibody, a molecularly imprinted polymer, an aptamer, or the like.
The invention also provides application of the detection method in detecting cancer cells, bacteria, macromolecular proteins and small molecular substances.
Preferably, the bacteria are pathogenic bacteria such as salmonella, the macromolecular protein is allergen, carcinoembryonic antigen and the like, and the micromolecular substance is residue of veterinary drugs and the like.
The assembly principle of the thermometer detection method for detecting cancer cells, bacteria and macromolecular proteins is based on the double-antibody sandwich principle, and the detection of small molecular substances is based on the competitive binding principle.
Advantageous effects
1. The invention proposes to use a thermometer as a carrier and build a sensitive and rapid thermometer detection method integrating detection carrier and signal generation based on the photothermal effect of a nano material. The method is used for the implementation scheme of sensitive and rapid detection of the salmonella, the whole detection time is about 2 hours, which is obviously shorter than the detection process of enzyme-linked immunity by about 3 hours. The detection sensitivity reaches 103CFU/mL is obviously superior to the detection sensitivity of the immune test strip.
2. Enzyme-linked immunosorbent assay or test strip quantitative detection requires an enzyme-linked immunosorbent assay (about 3.5 ten thousand yuan) or a card reader (about 2 ten thousand yuan), and the thermal imager for quantitative detection through the performance of the photothermal conversion material also has the cost of about 4 ten thousand yuan, so that the wide popularization of the traditional rapid quantitative detection method is limited by the high instrument cost. The invention utilizes the thermometer (about 60 yuan) to read the temperature change generated by the photothermal effect, greatly reduces the detection cost and is beneficial to the wide application of the detection method.
3. The invention takes the thermometer as a detection method carrier, can read the temperature change generated by the photothermal effect, integrates the detection carrier and the signal generation, and simplifies the operation steps of the whole method.
4. The invention realizes the photo-thermal signal conversion of graphene by using laser irradiation, directly records the temperature change by using a thermometer, establishes the relation between a target object to be detected and the temperature change and realizes the quantitative, rapid and sensitive detection of the target object to be detected.
5. The invention provides a coupling method of an antibody on the surface of a mercury head of a thermometer, which is characterized in that disordered valence bonds on the surface of a glass thermometer are treated, so that the surface of the glass thermometer is provided with stable active groups capable of being connected with a target recognition material (an antibody, a molecular imprinting polymer or an aptamer) as a carrier for a detection method.
Drawings
FIG. 1 is a schematic diagram of a process of capturing and detecting Salmonella typhimurium integrating detection carrier and signal generation based on graphene oxide photothermal effect.
FIG. 2 optimization of thermometer-conjugated antibody concentration.
Figure 3 optimization of graphene-conjugated antibody concentration.
FIG. 4 Standard Curve of Δ T vs. Salmonella typhimurium numbers.
FIG. 5 approach specificity.
Detailed Description
The invention is further illustrated by the following figures and examples. The following experiment of the invention illustrates the detection method of the invention by taking the detection of Salmonella typhimurium as an example, but is not limited to the detection of Salmonella typhimurium.
Example 1 method for coupling antibody on surface of mercury head of thermometer
1. Thermometer surface pretreatment
Firstly, cleaning the surface of a mercury head of a thermometer by using a cleaning agent to remove dirt on the surface, then cleaning by using deionized water, after the surface of the mercury head of the thermometer is dried to be anhydrous, placing the mercury head of the thermometer in 20mL of Piranha solution, carrying out ultrasonic treatment for 1h, cleaning by using a large amount of deionized water until the pH value of the surface of the mercury head is neutral, and drying for 2h at 65 ℃. After the thermometer was returned to room temperature, it was immersed in a 10% solution of 3-Aminopropyltriethoxysilane (APTES) in methanol, reacted at room temperature for 2 hours, and then the mercury head of the thermometer was washed 3 times with methanol to remove the remaining APTES, and the thermometer was placed in an oven to dry at 50 ℃ for 30 min.
2. Coupling of thermometer mercury head surface antibody
300 μ L of anti-Salmonella typhimurium antibody at a concentration of 3 μ g/mL was added to a polystyrene sample tube. And (3) inserting the treated thermometer mercury head into an anti-salmonella typhimurium antibody solution, incubating for 1.5h at 37 ℃, washing the mercury head for 3 times by using 0.01mol/L PBS (phosphate buffer solution), and washing away the unbound antibody to obtain the salmonella typhimurium antibody modified thermometer mercury head.
3. Optimization of thermometer surface antibody modification amount
Anti-salmonella typhimurium antibodies were modified to the surface of the mercurial head of the thermometer to specifically capture salmonella typhimurium. The amount of antibody coupled to the surface of the mercury head directly affects the efficiency of capture of the bacteria and thus the sensitivity of the detection method.
The invention modifies FITC-IgG on the surface of a mercury head of a thermometer to optimize the dosage of the antibody. Different concentrations of fluorescent antibody (0.1,0.5,1,2,3,4,5 mu g/mL) and a thermometer silver head are incubated for 1.5h at 37 ℃, the silver head is washed for 3 times by 0.01mol/L PBS (phosphate buffer solution), unbound antibody is washed away, and finally the fluorescent antibody is eluted by 1mol/L NaOH, and the amount of antibody modification on the surface of the thermometer is optimized by measuring the fluorescence intensity. As can be seen from FIG. 2, the fluorescence intensity gradually increased with the increase in the antibody concentration. When the concentration of the fluorescent antibody was 3. mu.g/mL, the fluorescence intensity of the eluted antibody reached almost the maximum value, and the increase in fluorescence intensity was insignificant with the increase in concentration. Thus, the optimized antibody concentration was 3. mu.g/mL.
Example 2 preparation method of immuno-graphene
1. Culture of Salmonella typhimurium
Picking a single colony of the salmonella typhimurium from the inclined plane, placing the single colony in an LB liquid culture medium, carrying out shaking culture at 37 ℃ for 12h, and then collecting a bacterial liquid. Before using the bacterial liquid, treating the salmonella typhimurium to reduce the interference of a culture medium on an experiment, adding a small amount of the bacterial liquid into a 1.5mL centrifugal tube, centrifuging for 5min at 3000r/min, discarding the supernatant, redissolving the precipitate by using a PBS solution, centrifuging again, repeating the steps for 3 times, then dissolving the precipitate by using the PBS solution and using.
2. Preparation of immune graphene
Adding 1mL of anti-salmonella typhimurium antibody with the concentration of 10 mug/mL into 1mL of graphene oxide solution with the concentration of 200 mug/mL, carrying out shake reaction at room temperature for 6h to obtain a compound of antibody graphene, namely immune graphene, and storing at 4 ℃.
3. Optimization of concentration of graphene coupled antibody
The graphene captures bacteria through the coupled antibody so as to be combined to the surface of the mercury head of the thermometer, the amount of the graphene coupled antibody influences the amount of the graphene connected to the surface of the mercury head, and finally influences the temperature rise effect, so that the sensitivity of the detection method is influenced, and therefore the concentration of the antibody incubated with the graphene needs to be optimized.
And incubating the graphene with FITC-IgG with different concentrations, centrifuging, and optimizing the dosage of the antibody by detecting the fluorescence intensity of the compound. The fixed graphene (2mg/mL,150 μ L) is incubated with FITC-IgG (0.5, 1, 5, 10, 20 μ g/mL) with different concentrations at 37 ℃ for 2h, then the mixture is centrifuged at 11200 Xg for 10min, the supernatant is discarded, the mixture is resuspended in 0.01mol/L PBS and then centrifuged again, the operation is repeated for 2 times, and finally the fluorescence intensity of the compound is detected. As can be seen from FIG. 3, the fluorescence intensity of the antibody gradually increased as the concentration of the antibody increased. When the antibody concentration was 10. mu.g/mL, the fluorescence intensity almost reached the maximum value, and the increase in fluorescence intensity was insignificant as the antibody concentration increased. Therefore, 10. mu.g/mL was selected as the optimized antibody concentration.
Example 3 method for capturing and detecting Salmonella typhimurium
The prepared thermometer with the surface modified with the anti-salmonella typhimurium antibody is inserted into a salmonella solution, incubated at 37 ℃ for 2h, washed with 0.01mol/L PBS for 3 times, and washed away from unbound bacteria. The mercury tip capturing Salmonella typhimurium was then inserted into the probe and incubated at 37 ℃ for 2h, followed by 3 washes of the thermometer with PBS. After it is dried, the mercury head is irradiated with laser, and the temperature change before and after irradiation is directly read by a thermometer. A standard curve was established using the temperature rise (. DELTA.T, equation 1) and the number of bacteria.
Δ T- Δ T1- Δ T0 formula 1
Δ T, temperature rise value
Δ T0, temperature rise before and after laser irradiation of blank (no bacteria added) samples.
Δ T1, temperature increase value before and after laser irradiation after sample addition.
In order to eliminate the influence of the graphene non-specifically adsorbed to the surface of the mercury head, the temperature increase value (Δ T0) before and after laser irradiation of the blank (no bacteria added) sample should be subtracted from the temperature increase value (Δ T1) before and after laser irradiation after the sample is added.
The laser is used to irradiate the mercury head part of the thermometer, the temperature change is obtained by reading the thermometer, the relationship between the temperature rise value △ T and the bacterial number is established by utilizing the photothermal effect of the graphene, and as can be seen from figure 4, the concentration of the salmonella typhimurium is 103～108Has good linear correlation in the CFU/mL range, and the minimum detection limit of the established detection method is 103CFU/mL。
The thermometer (shown in figure 1) which is prepared by the optimization experiment and based on the graphene oxide photothermal effect and integrates the detection carrier and the signal generation, can be used for rapidly detecting the salmonella typhimurium.
Example 4 recovery rate investigation based on thermometer detection method
After the mercury head surface of the thermometer prepared by optimization is modified with the anti-salmonella typhimurium antibody, the anti-salmonella typhimurium antibody is respectively mixed with the salmonella typhimurium (10)3,105,108CFU/mL, 300. mu.L) was incubated at 37 ℃ for 0.5h, and then the thermometer was washed 3 times with 0.01mol/L PBS to wash away unbound bacteria. The thermometer capturing the bacteria was incubated with the immuno-graphene complexes for 1h at 37 ℃, the thermometer was rinsed 3 times with PBS, and unbound graphene was washed away. After the mercury head surface is completely dried, the graphene area bonded on the thermometer is irradiated with 808nm laser, and the temperature change can be recorded by the thermometer. And (4) bringing the temperature change value into a standard curve to obtain a recovery rate of 95-112% corresponding to the concentration, and meeting the detection requirement.
Example 5 Selective detection Observation based on thermometer detection method
After the anti-salmonella typhimurium antibody is modified on the surface of the mercury head of the thermometer prepared by optimization, the anti-salmonella typhimurium antibody, the escherichia coli O157H 7 and staphylococcus aureus (1 multiplied by 10) are respectively added8CFU/mL, 300. mu.L) was incubated at 37 ℃ for 0.5h, the thermometer was rinsed 3 times with 0.01mol/L PBS, and unbound bacteria were washed away. The thermometer for capturing bacteria is respectively incubated with the immune graphene complex for 1h at 37 ℃, the thermometer is washed 3 times by PBS, and unbound graphene is washed away. Head for holding mercuryAs can be seen from FIG. 5, △ T generated by laser irradiation is significantly higher than Escherichia coli and Staphylococcus aureus when only Salmonella typhimurium is added, thus proving that the specificity of the method is better.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive faculty, based on the technical solutions of the present invention.
Claims (21)
1. A thermometer detection method integrating detection carrier and signal generation based on graphene oxide photothermal effect is characterized by comprising the following steps:
step (1): thermometer mercury head surface coupling target analyte antibody
Firstly, pretreating the surface of a mercury head of a thermometer, inserting the treated mercury head of the thermometer into an antibody solution for incubation, and cleaning;
step (2): preparation of immune graphene
Adding a target substance antibody to be detected into a graphene oxide solution for reaction to obtain an antibody-graphene compound, namely immune graphene;
and (3): detecting a target object to be detected;
the specific operation method of the step (3) comprises the following steps:
inserting the thermometer with the surface coupled with the antibody of the target object to be detected in the step (1) into the solution of the target object to be detected for incubation, washing the thermometer by using a buffer solution, and washing away the unbound object to be detected; then inserting the mercury head capturing the target substance to be detected into an immune graphene solution for incubation, and washing the thermometer with a buffer solution; after the mercury is dried, irradiating the position of the mercury head by laser, and directly reading the temperature change before and after irradiation by a thermometer; and establishing a standard curve by using the temperature rise value and the number of the target objects to be measured.
2. The graphene oxide photothermal effect based thermometer detection method integrating detection carrier and signal generation as a whole as claimed in claim 1, wherein the specific operation method of step (1) is as follows:
step 1.1 thermometer surface pretreatment
Cleaning the surface of the mercury head of the thermometer by using a cleaning agent, cleaning by using deionized water, drying until no water exists, placing the mercury head of the thermometer in a Piranha solution for ultrasonic treatment, cleaning by using a large amount of deionized water until the pH value of the surface of the mercury head is neutral, and drying; after the temperature of the thermometer is returned to the room temperature, the thermometer is immersed into APTES methanol solution for reaction; then washing the mercury head of the thermometer by using methanol, washing away residual APTES, and placing the thermometer in an oven for drying;
step 1.2 coupling of the antibody to the surface of the Mercury head of the thermometer
Adding the target substance antibody to be detected into a polystyrene sample tube, inserting the thermometer silver head treated in the step (1) into an antibody solution for incubation, washing the silver head with a buffer solution, and washing away the unbound antibody.
3. The graphene oxide photothermal effect based thermometer detection method integrating detection carrier and signal generation is characterized in that in the step 1.1, the ultrasonic time is 0.5-1.5 h; drying the mercury head of the thermometer at the temperature of 60-70 ℃ for 1.5-2.5 h after ultrasonic cleaning; the reaction conditions are as follows: reacting at room temperature for 2-2.5 h; drying the thermometer in an oven for 25-35 min at the temperature of 50-60 ℃; the volume of the Piranha solution is 20-25 mL; the mass fraction of the APTES methanol solution is 10 percent; the frequency of washing the mercury head of the thermometer by methanol is 2-3 times.
4. The graphene oxide photothermal effect based integrated detection carrier and signal generation thermometer detection method according to claim 3, wherein in step 1.1, the ultrasonic time is 1 h.
5. The graphene oxide photothermal effect based thermometer detection method integrating detection carrier and signal generation as a whole according to claim 3, wherein in the step 1.1, the drying condition after the ultrasonic cleaning of the mercury head of the thermometer is 65 ℃ for 2 h.
6. The graphene oxide photothermal effect based thermometer detection method integrating detection carrier and signal generation as a whole according to claim 3, wherein in the step 1.1, the reaction conditions are as follows: the reaction time is 2h at room temperature.
7. The graphene oxide photothermal effect based thermometer detection method integrating detection carrier and signal generation as a whole according to claim 3, wherein in the step 1.1, the thermometer is dried at 50 ℃ for 30min in an oven drying condition.
8. The method for detecting the thermometer integrating the detection carrier and the signal generation based on the graphene oxide photothermal effect according to claim 2, wherein in the step 1.2, the amount of the target analyte antibody is 300 to 350 μ L, and the concentration is 3 μ g/mL; incubating for 1-2 h at 35-40 ℃; the buffer solution is 0.01mol/L PBS, and the washing is carried out for 2-3 times.
9. The graphene oxide photothermal effect based integrated detection carrier and signal generation thermometer detection method according to claim 8, wherein in step 1.2, the incubation condition is 37 ℃ for 1.5 h.
10. The graphene oxide photothermal effect based thermometer detection method integrating detection carrier and signal generation as a whole according to claim 1, wherein the reaction condition in step (2) is room temperature shaking reaction for 5-7 h; the volume ratio of the target analyte antibody to the graphene oxide solution is 1: 1, the concentration of the target object antibody is 10 mug/mL, and the concentration of the graphene oxide solution is 200 mug/mL; the obtained immune graphene is stored at 4 ℃.
11. The method for detecting a thermometer integrating detection carrier and signal generation based on graphene oxide photothermal effect according to claim 10, wherein the reaction condition in step (2) is room temperature shaking reaction for 6 h.
12. The graphene oxide photothermal effect based thermometer detection method integrating detection carrier and signal generation is characterized in that in the step (3), the incubation in the target analyte solution is performed under the condition of 35-40 ℃ for 1.5-2.5 h; incubating for 1.5-2.5 h at 35-40 ℃ in the immune graphene solution; the buffer solution is 0.01mol/L PBS, and the washing is carried out for 3-4 times.
13. The method for detecting a thermometer integrating detection carrier and signal generation based on graphene oxide photothermal effect according to claim 12, wherein in the step (3), the incubation condition in the target analyte solution is 37 ℃ for 2 h.
14. The graphene oxide photothermal effect based integrated detection carrier and signal generation thermometer detection method according to claim 12, wherein in step (3), the incubation condition in the immune graphene solution is 37 ℃ for 2 h.
15. The method for detecting a thermometer integrating detection carrier and signal generation based on graphene oxide photothermal effect according to claim 1, wherein the analyte is cancer cell, bacteria, macromolecular protein and small molecular substance; the antibody can be replaced by a molecular imprinting polymer and a nucleic acid aptamer; the thermometer is a thermometer or a temperature sensor with various models; the graphene can be replaced by other materials with a photo-thermal effect.
16. The graphene oxide photothermal effect based thermometer detection method integrating detection carrier and signal generation as a whole as claimed in claim 15, wherein the bacteria is salmonella, the macromolecular proteins are allergens and carcinoembryonic antigens, and the small molecular substances are residues of veterinary drugs.
17. A coupling method of an antibody on the surface of a mercury head of a thermometer is characterized by comprising the following steps:
step (1) surface pretreatment of thermometer
Cleaning the surface of the mercury head of the thermometer by using a cleaning agent to remove dirt on the surface, then cleaning by using deionized water, drying until no water exists, putting the mercury head of the thermometer into Piranha solution for ultrasonic treatment, cleaning by using deionized water until the pH value of the surface of the mercury head is neutral, and drying; after the temperature of the thermometer is returned to the room temperature, the thermometer is immersed into APTES methanol solution for reaction; then washing the mercury head of the thermometer by using methanol, washing away residual APTES, and placing the thermometer in an oven for drying;
step (2) coupling of thermometer mercury head surface antibody
Adding the target substance antibody to be detected into a polystyrene sample tube, inserting the thermometer silver head treated in the step (1) into an antibody solution for incubation, washing the silver head with a buffer solution, and washing away the unbound antibody;
in the step (1), the ultrasonic time is 0.5-1.5 h; drying the mercury head of the thermometer at the temperature of 60-70 ℃ for 1.5-2.5 h after ultrasonic cleaning; the reaction conditions are as follows: reacting at room temperature for 2-2.5 h; drying the thermometer in an oven for 25-35 min at the temperature of 50-60 ℃; the volume of the Piranha solution is 20-25 mL; the mass fraction of the APTES methanol solution is 10 percent; the frequency of washing the mercury head of the thermometer by methanol is 3-4 times;
in the step (2), the dosage of the target to-be-detected object antibody is 300-350 mu L, and the concentration is 3 mu g/mL; incubating for 1-2 h at 35-40 ℃, washing for 2-3 times by using 0.01mol/L PBS as a buffer solution.
18. The method for coupling the antibody on the surface of the mercury head of the thermometer according to claim 17, wherein in the step (1), the drying condition after the ultrasonic cleaning of the mercury head of the thermometer is 65 ℃ for 2 h.
19. The method for coupling an antibody on the surface of a mercury head of a thermometer according to claim 17, wherein in the step (1), the reaction conditions are as follows: the reaction time is 2h at room temperature.
20. The method for coupling the antibody on the mercury head surface of the thermometer according to claim 17, wherein in the step (1), the thermometer is dried in an oven at 50 ℃ for 30 min.
21. The method for coupling an antibody to the mercury head surface of a thermometer according to claim 17, wherein in the step (2), the incubation is performed under the incubation condition of 37 ℃ for 1.5 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810725536.8A CN109060178B (en) | 2018-07-04 | 2018-07-04 | Thermometer rapid detection method integrating detection carrier and signal generation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810725536.8A CN109060178B (en) | 2018-07-04 | 2018-07-04 | Thermometer rapid detection method integrating detection carrier and signal generation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109060178A CN109060178A (en) | 2018-12-21 |
| CN109060178B true CN109060178B (en) | 2020-05-05 |
Family
ID=64818716
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810725536.8A Active CN109060178B (en) | 2018-07-04 | 2018-07-04 | Thermometer rapid detection method integrating detection carrier and signal generation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109060178B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110095602A (en) * | 2019-05-24 | 2019-08-06 | 山东师范大学 | A kind of cell detection kit based on graphene oxide, method and application |
| CN111521783A (en) * | 2020-03-26 | 2020-08-11 | 军事科学院军事医学研究院军事兽医研究所 | Nanoflower sensor system and preparation method and application thereof |
| CN113899701A (en) * | 2021-09-30 | 2022-01-07 | 合肥工业大学 | Nano sensor and preparation, application and test method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1475805A (en) * | 2002-08-15 | 2004-02-18 | 陕西西大北美基因股份有限公司 | Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere |
| CN103893764A (en) * | 2012-12-25 | 2014-07-02 | 国家纳米科学中心 | Drug carrier, preparation method thereof, pharmaceutical composition made from drug carrier, and applications of drug carrier and pharmaceutical composition |
| CN104800845A (en) * | 2015-01-28 | 2015-07-29 | 东华大学 | Preparation method of hepatoma carcinoma cell targeted molybdenum disulfide drug-loaded nano tablets |
| CN108195768A (en) * | 2017-12-25 | 2018-06-22 | 山东省圣泉生物质石墨烯研究院 | A kind of method that determinand light thermal property is characterized under the conditions of liquid |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10094793B2 (en) * | 2015-09-19 | 2018-10-09 | The Board Of Regents Of The University Of Texas System | Nanomaterial-based photothermal immunosensing for quantitative detection of disease biomarkers |
-
2018
- 2018-07-04 CN CN201810725536.8A patent/CN109060178B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1475805A (en) * | 2002-08-15 | 2004-02-18 | 陕西西大北美基因股份有限公司 | Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere |
| CN103893764A (en) * | 2012-12-25 | 2014-07-02 | 国家纳米科学中心 | Drug carrier, preparation method thereof, pharmaceutical composition made from drug carrier, and applications of drug carrier and pharmaceutical composition |
| CN104800845A (en) * | 2015-01-28 | 2015-07-29 | 东华大学 | Preparation method of hepatoma carcinoma cell targeted molybdenum disulfide drug-loaded nano tablets |
| CN108195768A (en) * | 2017-12-25 | 2018-06-22 | 山东省圣泉生物质石墨烯研究院 | A kind of method that determinand light thermal property is characterized under the conditions of liquid |
Non-Patent Citations (1)
| Title |
|---|
| 基于石墨烯及室温离子液体的弓形虫IgM抗体免疫传感器的研制;陈中平;《分析仪器》;20131231(第1期);第63-68页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109060178A (en) | 2018-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Simultaneous electrochemical immunoassay of three liver cancer biomarkers using distinguishable redox probes as signal tags and gold nanoparticles coated carbon nanotubes as signal enhancers | |
| CN109060178B (en) | Thermometer rapid detection method integrating detection carrier and signal generation | |
| CN103116023B (en) | ECL (electrochemiluminescence) immunosensor for detecting tumor markers and preparation method and applications thereof | |
| CN106018806B (en) | One kind is based on Au Fe3O4The preparation method and application for the interlayer type immunosensor that@Pd ZIF 8 are marked | |
| CN104391117B (en) | Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe | |
| JP2004317520A (en) | Taxonomic identification of pathogenic microorganism and toxic protein of the pathogenic microorganism | |
| CN107843629B (en) | A kind of preparation method detecting A549 tumour cell electrochemical sensor working electrode | |
| CN105675697A (en) | Construction method of nanoprobe C60 based electrochemical immunosensor for carcino-embryonic antigens | |
| CN104833718B (en) | A kind of preparation method of pH release type immunosensor and application | |
| CN110308286B (en) | Enhanced thyroglobulin electrochemiluminescence immunosensor based on photothermal release signals | |
| CN105319254A (en) | Preparation and application of electrochemical immunosensor based on Pt/PdCu-three-dimensional graphene markers | |
| CN105241939A (en) | Preparation method for immunosensor based on gold/silver core-shell magnetic graphene adsorption cadmium ion and application | |
| CN104880456A (en) | Preparation method and application of electrochemiluminescence immunosensor constructed on basis of GO/MWCNTs-COOH/Au @ CeO2 | |
| CN104931698B (en) | The preparation method of a kind of stomach cancer marker gold nanoclusters Electrochemiluminescsensor sensor based on NP-NiGdAu and application | |
| CN108226461A (en) | Electrochemical luminescence immunosensor based on CdZnTeS quantum dots and its preparation method and application | |
| CN108918853B (en) | Pd @ Ag @ CeO2Preparation method and application of labeled immunosensor | |
| CN114324863B (en) | Staphylococcus aureus toxin detection biological probe based on optical/electric double signal mode, and immunosensor and detection method thereof | |
| CN110441528B (en) | Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor | |
| CN101498719A (en) | Production method for enzyme functionalized nano immunity marker and use thereof | |
| CN110031527B (en) | Double-reading biosensor for human thyroglobulin | |
| CN103869063B (en) | The preparation method of strong anti-matrix interference type bisphenol-A up-conversion fluorescence chromatograph test strip | |
| CN107525835A (en) | A kind of preparation method and application of the immunosensor of the phenolic resin micropore carbon ball of functionalization based on Au AgNPs | |
| CN107271519B (en) | A kind of preparation method and application of the immunosensor of the Sulfonated carbon nanotube based on load Rh@Pd nanodendrites | |
| CN104198563B (en) | Preparation method and the application of lead ion gold-supported magnetic multi-wall carbon nano-tube tube sensor | |
| CN106323952B (en) | A kind of preparation method and application of the electroluminescent immunosensor based on double coreagent amplified signals |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |