CN115248310A - Immunochromatography detection method based on Janus platinum nanomotor and application - Google Patents
Immunochromatography detection method based on Janus platinum nanomotor and application Download PDFInfo
- Publication number
- CN115248310A CN115248310A CN202210576462.2A CN202210576462A CN115248310A CN 115248310 A CN115248310 A CN 115248310A CN 202210576462 A CN202210576462 A CN 202210576462A CN 115248310 A CN115248310 A CN 115248310A
- Authority
- CN
- China
- Prior art keywords
- solution
- janus
- platinum
- ultrasonic
- nanomotor
- 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.)
- Granted
Links
Images
Classifications
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of immunodetection, and discloses an immunochromatography detection method based on a Janus platinum nano motor and application thereof, wherein the Janus platinum nano motor with 1mg/mL-5mg/mL carboxyl functionalization is activated for 1-2 hours by using 1mg/mL-5mg/mL EDC and 1mg/mL-5mg/mLNHS, and then is coupled for 4-6 hours by using 0.1mg-1mg target protein; blocking the coupled product for 1-2 hours by using 10% BSA solution after coupling; and then uniformly spraying the gold on glass fiber by using an XYZ three-dimensional film-scribing gold spraying instrument, and finally detecting the target antigen by using a sample solution added with 1-10wt% of hydrogen peroxide. According to the invention, the combination efficiency of the antigen and the antibody can be increased by means of the special motion state of the nano motor in the solution, so that the detection capability of the immunochromatographic test strip on the antigen is improved.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to an immunochromatography detection method based on a Janus platinum nano motor and application thereof.
Background
At present, the immunochromatography technology is mostly based on a colloidal gold probe or a fluorescent probe, and has the advantages of low detection cost and convenient use. However, most of the methods for detecting the signal of the colloidal gold probe realize qualitative detection by a colorimetric method, i.e., the intensity of color is distinguished by naked eyes, so that the problems of low sensitivity and difficulty in accurate quantification exist. Although the fluorescent probe can realize quantitative detection, the luminescence type of the fluorescent probe mostly belongs to down-conversion fluorescence, namely, high-energy photons are used for exciting low-energy photons so as to emit fluorescence, and the detection method has the defects of being easily interfered by biological endogenous fluorescence and having poor signal stability. In addition, the existing micro-nano probes detect the immune markers in a passive mode, and the probes passively diffuse forwards through capillary action, so that the contact time and the contact area between antigen and antibody are limited, and the detection sensitivity is influenced. Therefore, in order to solve the problems of low stability, poor sensitivity and the like in the prior art, an immunochromatography detection technology based on an active mode needs to be developed. The Janus nano motor is used as a micro-nano device capable of autonomously moving, bubbles are generated by oxidation-reduction reaction initiated by chemical fuel (such as hydrogen peroxide), and then bubble-driven movement is achieved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an immunochromatography detection method based on a Janus platinum nano motor and application thereof.
The immunochromatography detection method based on the Janus platinum nano motor is realized by activating 1mg/mL-5mg/mL carboxyl functionalized Janus platinum nano motor with 1mg/mL-5mg/mL EDC and 1mg/mL-5mg/mL LNHS for 1-2 hours and then coupling the activated Janus platinum nano motor with 0.1mg-1mg target protein for 4-6 hours; blocking the coupled product for 1-2 hours by using 10% BSA solution after coupling; and then uniformly spraying the gold on glass fiber by using an XYZ three-dimensional film-scribing gold spraying instrument, and finally detecting the target antigen by using a sample solution added with 1-10wt% of hydrogen peroxide.
Further, the immunochromatography detection method based on the Janus platinum nanomotor specifically comprises the following steps:
taking a certain concentration of Janus platinum nano-motor microsphere solution coupled with carboxyl out of a refrigerator, and carrying out ultrasonic treatment on the solution by using an ultrasonic cell crusher, wherein the ultrasonic power is 180W, two seconds of ultrasonic treatment are temporarily stopped for one group, three groups are carried out in total, and the ultrasonic treatment time is 6 seconds; after the ultrasonic treatment is finished, putting the Janus platinum nano motor solution into a centrifuge tube, adding an MES solution into the centrifuge tube, pumping and uniformly mixing the solution by using a liquid-transferring gun, so that the concentration of the prepared Janus platinum nano motor solution is 1mg/mL-5mg/mL, and the volume of the solution is 1mL; then centrifuging by using a centrifuge, and finishing the cleaning of the Janus platinum nano motor microsphere solution in the step;
secondly, taking the EDC and the NHS out of the refrigerator during centrifugation for 5 minutes, and then respectively weighing 1mg/mL-5mg/mL EDC and 1mg/mL-5mg/mL by using an electronic balance; pouring the two weighed medicines into an empty centrifuge tube, adding MES solution into the empty centrifuge tube by using a pipette, pumping and uniformly mixing, and completing the preparation of the activating reagent;
taking out the centrifugal tube after the centrifugation is finished, sucking the supernatant in the centrifugal tube by using a liquid transfer gun and pumping the supernatant into a waste liquid cylinder for the centrifugal tube provided with the Janus platinum nano motor, adding 600uL of MES solution into the centrifugal tube after the supernatant is completely taken away, and carrying out ultrasound by using a cell disruption ultrasonic machine; adding 400uL of MES solution mixed with EDC and NHS prepared in the second step after the ultrasonic treatment is finished, fixing the centrifugal tube in a rotary blending machine for activation for 1-2 hours, setting the rotating speed to be 15, and finishing the activation of the Janus platinum nanomotor microsphere solution in the step;
fourthly, centrifuging by using a centrifugal machine after activation is finished, wherein the rotating speed of the centrifugal machine is 12000rpm, the working time is 6min, and the working temperature is 4 ℃; taking out the centrifugal tube after the centrifugation is finished, sucking the supernatant in the centrifugal tube by using a pipette into a waste liquid cylinder for the centrifugal tube provided with the Janus platinum nano motor, adding 1mL of PBS solution into the centrifugal tube after the supernatant is completely taken away, and carrying out ultrasound by using a cell disruption ultrasonic machine, wherein the step can be used for cleaning redundant activating agent in the solution;
fifthly, adding 0.1-1 mg of antibody for coating into the Janus platinum nano motor solution by using a pipette gun, and then fixing the centrifuge tube on a rotating disc to rotate for labeling for 4-6 hours, wherein the step can finish the labeling coupling of the target antibody;
sixthly, centrifuging by using a centrifugal machine after marking is finished, taking out the centrifugal tube after centrifugation is finished, sucking supernatant in the centrifugal tube by using a liquid transfer gun to the centrifugal tube provided with the Janus platinum nano motor, pumping the supernatant into a waste liquid cylinder, adding a PBS (phosphate buffer solution) solution into the centrifugal tube after the supernatant is completely taken away, and performing ultrasound by using a cell disruption ultrasonic machine; the ultrasonic power is 180W, 2 seconds of ultrasonic pause are set as a group, 2 groups are carried out totally, the ultrasonic time is 4 seconds in total, the ultrasonic environment is an ice bath, and the step can be used for cleaning the antibody which is not coupled in the solution;
a seventh step of adding a 10% BSA solution to the Janus platinum nanomotor solution by using a pipette gun, and then rotating the centrifuge tube on a rotating disk for blocking for 1-2 hours, wherein the carboxyl sites of the Janus platinum nanomotor microspheres which are not coupled with the antibody protein can be blocked;
step eight, centrifuging by using a centrifuge after sealing is finished, wherein the rotating speed of the centrifuge is 12000rpm, the working time is 6min, and the working temperature is 4 ℃; taking out the centrifugal tube after the centrifugation is finished, sucking the supernatant in the centrifugal tube by a pipette gun and pumping the supernatant into a waste liquid cylinder for a Janus platinum nano motor, adding 1mL of 1-percent BSA solution into the centrifugal tube after the supernatant is completely removed, and carrying out ultrasound by using a cell disruption ultrasonic machine; the ultrasonic power is 180W, the ultrasonic is suspended for 2 seconds to form a group, 2 groups are carried out totally, the ultrasonic time is 4 seconds, the ultrasonic environment is an ice bath, the antibody coupling is completed after the ultrasonic is finished, and the step can ensure that the coupled antibody-microsphere solution can be stably stored for a long time;
ninthly, spraying gold on the glass fiber treated by the glass fiber treatment solution by the Janus platinum nanomotor solution coupled with the antibody according to the spraying amount of 2.0 uL/cm; after the gold spraying is finished, placing the glass fiber in a drying oven at 37 ℃ for drying for 24 hours, wherein the Janus platinum nano motor probe coupled with the antibody can be fixed on the glass fiber;
tenth step, pasting absorbent paper on the uppermost end of the PVC bottom card, and pasting an NC film on the middle part; diluting the antibody for scribing to 1mg/mL by using an antibody diluent, scribing a detection line on an NC membrane according to the spraying amount of 1uL/cm, diluting the secondary antibody to 1mg/mL by using the antibody diluent, and scribing a control line on the NC membrane according to the spraying amount of 1 uL/cm; after the scribing is finished, the test card is placed into a drying oven at 37 ℃ to be dried for 24 hours, and the protein antibody can be fixed on an NC membrane in the step;
step eleven, after drying, sticking the glass fiber to the lowest end of the PVC bottom card, and cutting the PVC bottom card into test strips with the width of 4mm by using a high-speed cutting machine;
and step ten, adding a hydrogen peroxide solution into the sample solution to enable the mass fraction of the hydrogen peroxide solution to be 1-10wt%, and dropwise adding 90uL of the hydrogen peroxide solution to a test strip for antigen detection, wherein the Janus platinum nano motor can autonomously move on an NC membrane in the step, so that the effects of enhancing the detection sensitivity and stability are achieved.
Further, after the first-step ultrasound is finished, 0.4mL of Janus platinum nanomotor solution is taken to enter a 1.5mL centrifuge tube, 0.6mL of MES solution is added into the centrifuge tube, and the mixture is pumped and uniformly mixed by a pipette, so that the concentration of the configured Janus platinum nanomotor solution is 1-5mg/mL, and the volume of the solution is 1mL; and then centrifuging by using a centrifuge, setting the rotation speed of the centrifuge to 12000rpm, working time to 6min and working temperature to 4 ℃.
And further, in the second step, the two weighed medicines are poured into a 1.5mL empty centrifuge tube, a 400uL MES solution is added into the empty centrifuge tube by using a pipette gun, and the mixture is pumped and uniformly mixed.
Further, the ultrasonic power of the third step is 180W, 2 seconds of ultrasonic pause are set as a group, 2 groups are carried out totally, and the ultrasonic time is 4 seconds in total.
Further, the ultrasonic power of the fourth step is 180W, 2 seconds of ultrasonic pause are one group, 2 groups are performed totally, and the ultrasonic time length is 4 seconds in total.
Further, the fifth step adds 0.25mg of the antibody for coating to the solution of the Janus platinum nanomotor using a pipette gun, and then the centrifuge tube is fixed on a rotating disk and rotated for 4 to 6 hours for labeling.
And further, after marking in the sixth step is finished, centrifuging by using a centrifuge, setting the rotating speed of the centrifuge to be 12000rpm, working time to be 6min and working temperature to be 4 ℃, taking out the centrifuge tube after centrifuging, taking supernatant in the centrifuge tube into a waste liquid cylinder for the centrifuge tube with the Janus platinum nanometer motor, and adding 1mL of PBS solution into the centrifuge tube after all the supernatant is taken away.
Further, the seventh step is to add 100uL of 10% BSA solution to the Janus platinum nanomotor solution using a pipette gun, and then to block the tube by fixing it on a rotating disk and rotating it for 1 to 2 hours.
The invention also aims to provide a method for improving the detection capability of an immunochromatographic test strip on an antigen, which comprises the immunochromatographic detection method based on the Janus platinum nanomotor.
In combination with the technical solutions and the technical problems to be solved, please analyze the advantages and positive effects of the technical solutions to be protected in the present invention from the following aspects:
first, aiming at the technical problems existing in the prior art and the difficulty in solving the problems, the technical problems to be solved by the technical scheme of the present invention are closely combined with the technical scheme to be protected and the results and data in the research and development process, and some creative technical effects brought after the problems are solved are analyzed in detail and deeply. The specific description is as follows: the autonomous movement characteristic of the Janus platinum nanomotor is utilized to be used as a probe to couple immune protein (antigen, antibody or aptamer), so that the immune protein can be stably attached to the Janus platinum nanomotor probe. In the detection process, hydrogen peroxide with a certain concentration is added into a sample solution, the automatic movement of the Janus platinum nanometer motor probe on the NC membrane is realized by utilizing bubble-driven movement, the combination efficiency of the antigen and the antibody is improved, so that the immunochromatography detection based on an active mode is completed, and finally, the detection sensitivity and the stability are obviously improved.
Secondly, considering the technical scheme as a whole or from the perspective of products, the technical effect and advantages of the technical scheme to be protected by the invention are specifically described as follows: the invention provides an immunochromatography detection method based on Janus platinum nano motor by combining Janus nano motor as a probe with an immunochromatography technology, and the combination efficiency of antigen and antibody is increased by the characteristic that the motor can move autonomously, so that the detection capability of the immunochromatography test strip on antigen is improved.
Third, as inventive supplementary proof of the claims of the present invention, there are several important aspects as follows:
the expected income and commercial value after the technical scheme of the invention is converted are as follows:
after the technical scheme of the invention is successfully converted, iteration or upgrading of the existing partial immunochromatography technology is expected. Due to low preparation cost and higher detection sensitivity and detection stability, the invention can be widely applied to medical institutions such as drugstores, clinics, hospitals and the like, and realizes the rapid, accurate and quantitative detection of various immune indexes of a plurality of users. The invention is expected to improve the market share of the immunochromatographic test paper products in China and reduce the medical cost.
Drawings
FIG. 1 is a flow chart of an immunochromatographic assay method based on a Janus platinum nanomotor according to an embodiment of the present invention;
FIG. 2 is a linear detection diagram of pepsinogen I by a Janus platinum nano-motor based immunochromatographic method provided by the embodiment of the invention;
FIG. 3 is a linear detection diagram of pepsinogen II by a Janus platinum nano-motor based immunochromatographic method provided by an embodiment of the invention;
FIG. 4 is a specificity test chart of the Janus platinum nanomotor based immunochromatography method provided by the embodiment of the invention;
FIG. 5 is a specificity test of the Janus platinum nanomotor based immunochromatography method provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Illustrative embodiments are explained. This section is an explanatory embodiment expanding on the claims so as to fully understand how the present invention is embodied by those skilled in the art.
As shown in fig. 1, the immunochromatographic detection method based on a Janus platinum nanomotor provided by the embodiment of the present invention includes the following steps:
s101: activating the 1mg/mL-5mg/mL carboxyl functionalized Janus platinum nanomotor with 1mg/mL-5mg/mL EDC and 1mg/mL-5mg/mL LNHS for 1-2 hours;
s102: coupling the protein with 0.1-1 mg of target protein for 4-6 hours; blocking the conjugated protein with 10% BSA solution for 1-2 hours after coupling;
s103: then uniformly spraying the glass fiber with an XYZ three-dimensional film-scribing gold spraying instrument;
s104: finally, the target antigen is detected by using a sample solution added with 1-10wt% of hydrogen peroxide.
The immunochromatography detection method based on the Janus platinum nanomotor provided by the embodiment of the invention specifically comprises the following steps:
taking a certain concentration of Janus platinum nano-motor microsphere solution coupled with carboxyl out of a refrigerator, and carrying out ultrasonic treatment on the solution by using an ultrasonic cell crusher, wherein the ultrasonic power is 180W, two seconds of ultrasonic treatment are temporarily stopped for one group, three groups are carried out in total, and the ultrasonic treatment time is 6 seconds; after the ultrasonic treatment is finished, putting the Janus platinum nano motor solution into a centrifuge tube, adding an MES solution into the centrifuge tube, pumping and uniformly mixing the solution by using a liquid-transferring gun, so that the concentration of the prepared Janus platinum nano motor solution is 1mg/mL-5mg/mL, and the volume of the solution is 1mL; then centrifuging by using a centrifuge, and finishing the cleaning of the Janus platinum nano motor microsphere solution in the step;
secondly, taking the EDC and the NHS out of the refrigerator during centrifugation for 5 minutes, and then respectively weighing 1mg/mL-5mg/mL EDC and 1mg/mL-5mg/mL by using an electronic balance; pouring the two weighed medicines into an empty centrifuge tube, adding MES solution into the empty centrifuge tube by using a pipette, pumping and uniformly mixing, and completing the preparation of the activating reagent;
taking out the centrifugal tube after the centrifugation is finished, sucking the supernatant in the centrifugal tube by using a liquid transfer gun and pumping the supernatant into a waste liquid cylinder for the centrifugal tube provided with the Janus platinum nano motor, adding 600uL of MES solution into the centrifugal tube after the supernatant is completely taken away, and carrying out ultrasound by using a cell disruption ultrasonic machine; adding 400uL of MES solution mixed with EDC and NHS prepared in the second step after the ultrasonic treatment is finished, fixing the centrifugal tube in a rotary blending machine for activation for 1-2 hours, setting the rotating speed to be 15, and finishing the activation of the Janus platinum nanomotor microsphere solution in the step;
fourthly, centrifuging by using a centrifugal machine after activation is finished, wherein the rotating speed of the centrifugal machine is 12000rpm, the working time is 6min, and the working temperature is 4 ℃; taking out the centrifugal tube after the centrifugation is finished, sucking supernatant in the centrifugal tube by using a pipette gun and pumping the supernatant into a waste liquid cylinder for the centrifugal tube provided with the Janus platinum nano motor, adding 1mL of PBS solution into the centrifugal tube after the supernatant is completely taken away, and performing ultrasound by using a cell disruption ultrasonic machine, wherein the step can be used for cleaning redundant activating agent in the solution;
fifthly, adding 0.1-1 mg of antibody for coating into the Janus platinum nano motor solution by using a pipette, and then fixing the centrifuge tube on a rotating disc to rotate for labeling for 4-6 hours, wherein the step can finish labeling coupling of the target antibody;
sixthly, centrifuging by using a centrifugal machine after marking is finished, taking out the centrifugal tube after centrifugation is finished, sucking supernatant in the centrifugal tube by using a liquid transfer gun to the centrifugal tube provided with the Janus platinum nano motor, pumping the supernatant into a waste liquid cylinder, adding a PBS (phosphate buffer solution) solution into the centrifugal tube after the supernatant is completely taken away, and performing ultrasound by using a cell disruption ultrasonic machine; the ultrasonic power is 180W, 2 seconds of ultrasonic pause are set as a group, 2 groups are carried out totally, the ultrasonic time is 4 seconds in total, the ultrasonic environment is an ice bath, and the step can be used for cleaning the antibody which is not coupled in the solution;
a seventh step of adding a 10% BSA solution to the Janus platinum nanomotor solution by using a pipette gun, and then rotating the centrifuge tube on a rotating disk for blocking for 1-2 hours, wherein the carboxyl sites of the Janus platinum nanomotor microspheres which are not coupled with the antibody protein can be blocked;
step eight, centrifuging by using a centrifuge after sealing is finished, wherein the rotating speed of the centrifuge is 12000rpm, the working time is 6min, and the working temperature is 4 ℃; taking out the centrifuge tube after the centrifugation, sucking the supernatant in the centrifuge tube by a pipette gun for the Janus platinum nanometer motor, pumping the supernatant into a waste liquid cylinder, adding 1mL of 1% BSA solution into the centrifuge tube after the supernatant is completely removed, and performing ultrasound by using a cell disruption ultrasound machine; the ultrasonic power is 180W, 2 seconds of ultrasonic pause are one group, 2 groups are carried out totally, the ultrasonic time is 4 seconds in total, the ultrasonic environment is an ice bath, and the antibody coupling is completed after the ultrasonic is finished, so that the coupled antibody-microsphere solution can be stably stored for a long time;
ninthly, spraying gold on the glass fiber treated by the glass fiber treatment solution by the Janus platinum nanomotor solution coupled with the antibody according to the spraying amount of 2.0 uL/cm; after the gold spraying is finished, placing the glass fiber in a drying oven at 37 ℃ for drying for 24 hours, wherein the Janus platinum nano motor probe coupled with the antibody can be fixed on the glass fiber;
tenth step, pasting absorbent paper on the uppermost end of the PVC bottom card, and pasting an NC film on the middle part; diluting an antibody for scribing to 1mg/mL by using an antibody diluent, scribing a detection line on an NC membrane according to the spraying amount of 1uL/cm, diluting a secondary antibody to 1mg/mL by using the antibody diluent, and scribing a control line on the NC membrane according to the spraying amount of 1 uL/cm; after the scribing is finished, the test card is placed into a drying oven at 37 ℃ to be dried for 24 hours, and the protein antibody can be fixed on an NC membrane;
step eleven, after drying, sticking the glass fiber to the lowest end of the PVC bottom card, and cutting the PVC bottom card into test strips with the width of 4mm by using a high-speed cutting machine;
and step ten, adding a hydrogen peroxide solution into the sample solution to enable the mass fraction of the hydrogen peroxide solution to be 1-10wt%, and dropwise adding 90uL of the hydrogen peroxide solution to a test strip for antigen detection, wherein the Janus platinum nano motor can autonomously move on an NC membrane in the step, so that the effects of enhancing the detection sensitivity and stability are achieved.
Example 1
As shown in FIG. 2, the present invention detects pepsinogen I:
0.25mg of PG I-MAb 1 was used for coupling, 0.25mg of PG I-MAb 2 was used for scribing a detection line, and 1mg/mL of goat anti-rabbit IgG was used for scribing a quality control line, and test strips were prepared according to the above procedure. Using PG I antigen solution with hydrogen peroxide concentration of 5wt% as sample solution, and preparing PG I concentration gradient as follows: 0, 50, 10, 20, 50, 100, 500ng/mL. And (3) respectively taking 90uL of each concentration, dropwise adding the 90uL of each concentration into the test strip, and measuring and calculating the signal intensity of the detection line under different concentrations by using image processing software ImageJ after 10 minutes.
Example 2
As shown in FIG. 3, the present invention detects pepsinogen II:
0.25mg of PG II-MAb 1 is used for coupling, 0.25mg of PG II-MAb 2 is used for marking a detection line, 1mg/mL of goat anti-rabbit IgG is used for marking a quality control line, and the test strip is prepared according to the steps. Taking a PG II antigen solution with the hydrogen peroxide concentration of 5wt% as a sample solution, and preparing a PG II concentration gradient as follows: 0, 50, 10, 20, 50, 100, 500ng/mL. And (3) respectively dripping 90uL of each concentration into the test strip, and measuring and calculating the signal intensity of the detection line under different concentrations by using image processing software ImageJ after 10 minutes.
Example 3
As shown in fig. 4, the present invention verifies the specificity of the test pepsinogen:
0.25mg of PG I-MAb 1 and PG II-MAb 1 was used for coupling, 0.25mg of PG I-MAb 1 was used for scribing detection line 1 (T1) and PG II-MAb 2 was used for scribing detection line 2 (T1), and 1mg/mL of goat anti-rabbit IgG was used for scribing quality control line, and test strips were prepared according to the above procedure. A mixed antigen solution of PG I and PG II with hydrogen peroxide concentration of 5wt% was used as a sample solution, and the concentration was set to 20ng/mL. And f, respectively using CRP, SAA, PCT and MYO antigen solutions with hydrogen peroxide concentration of 5wt% as sample solutions, wherein the preparation concentrations are 20ng/mL. And taking 90uL of each antigen solution in decibels, dropwise adding the antigen solution into the test strip, and measuring and calculating the signal intensity of the detection line under different antigen sample solutions by using image processing software ImageJ after 10 minutes.
2. Application examples. In order to prove the creativity and the technical value of the technical scheme of the invention, the part is the application example of the technical scheme of the claims on specific products or related technologies.
By utilizing the technical scheme of the invention, the Janus platinum nanomotor probe is coupled with C-reactive protein (CRP) and Serum Amyloid A (SAA) respectively. After the steps of coupling, gold spraying and scribing, CRP antigen and SAA antigen in human serum are detected. The specific detection method is that 5wt% of hydrogen peroxide is added into 90 microliter of human serum sample, and the mixture is uniformly mixed and then is dripped on a test strip. At the same time, the sample solutions were added dropwise to the commercial CRP and SAA test strip products under the same conditions, and both groups reacted for 15 minutes. After the reaction is finished, the detection limits of the two groups of CRP test paper and SAA test paper are calculated, and the results show that the CRP detection limit of the invention is 0.05ng/mL, the sensitivity of the invention is improved by 50 percent compared with the commercial test paper, the SAA detection limit is 0.3 ng/mL, and the sensitivity of the invention is improved by 67 percent compared with the commercial test paper
3. Evidence of the relevant effects of the examples. The embodiment of the invention has some positive effects in the process of research and development or use, and indeed has great advantages compared with the prior art, and the following contents are described by combining data, charts and the like in the test process.
Pepsinogen i linear detection experiment:
experiment time: 2021.2.17
An experimental instrument: image J
Detection time: 10 minutes
The experimental scheme is as follows: and (3) establishing a linear relation of the signal intensity of the pepsinogen I concentration detection line by testing the signal intensity values of the detection line under different pepsinogen I concentrations, and calculating a linear detection range and a lowest detection limit.
The results of the experiment are shown in FIG. 2.
Pepsinogen II Linear detection experiment:
experiment time: 2021.2.17
An experimental instrument: image J
Detection time: 10 minutes
The experimental scheme is as follows: and (3) establishing a linear relation of the signal intensity of the pepsinogen II concentration detection line by testing the signal intensity values of the detection line under different pepsinogen II concentrations, and calculating a linear detection range and a lowest detection limit.
The results of the experiment are shown in FIG. 3.
Experiment 3
Specific experiment of pepsinogen detection:
experiment time: 2021.2.17
An experimental instrument: image J
Detection time: 10 minutes
The experimental scheme is as follows: the method verifies whether the antigen has good specificity by testing the signal intensity values of the detection lines under different antigen sample solutions.
The experimental results are shown in FIG. 4.
In conclusion, the Janus platinum nano motor can be used as an immunochromatography probe, so that the detection sensitivity of immunochromatography in the aspect of antigen detection is improved, and the immunochromatography probe has good development potential in the field of biological probes.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The immunochromatography detection method based on the Janus platinum nanomotor is characterized in that 1mg/mL-5mg/mL of carboxyl functionalized Janus platinum nanomotor is activated for 1-2 hours by using 1mg/mL-5mg/mL EDC and 1mg/mL-5mg/mL NHS, and then is coupled for 4-6 hours by using 0.1mg-1mg of target protein; blocking the conjugated protein with 10% BSA solution for 1-2 hours after coupling; and then uniformly spraying the gold on glass fiber by using an XYZ three-dimensional film-scribing gold spraying instrument, and finally detecting the target antigen by using a sample solution added with 1-10wt% of hydrogen peroxide.
2. The Janus platinum nanomotor-based immunochromatographic detection method of claim 1, wherein the Janus platinum nanomotor-based immunochromatographic detection method specifically comprises the following steps:
taking a certain concentration of Janus platinum nano-motor microsphere solution coupled with carboxyl out of a refrigerator, and carrying out ultrasonic treatment on the solution by using an ultrasonic cell crusher, wherein the ultrasonic power is 180W, two seconds of ultrasonic treatment are temporarily stopped for one group, three groups are carried out in total, and the ultrasonic treatment time is 6 seconds; after the ultrasonic treatment is finished, putting the Janus platinum nano motor solution into a centrifuge tube, adding an MES solution into the centrifuge tube, pumping and uniformly mixing the solution by using a liquid-transferring gun, so that the concentration of the prepared Janus platinum nano motor solution is 1mg/mL-5mg/mL, and the volume of the solution is 1mL; then centrifuging by a centrifuge;
secondly, taking the EDC and the NHS out of the refrigerator during centrifugation for re-warming for 5 minutes, and then weighing 1mg/mL-5mg/mL EDC and 1mg/mL-5mg/mL EDC respectively by using an electronic balance; pouring the weighed two medicines into an empty centrifuge tube, adding MES solution into the empty centrifuge tube by using a liquid transfer gun, pumping and uniformly mixing;
taking out the centrifugal tube after the centrifugation is finished, sucking the supernatant in the centrifugal tube by using a liquid transfer gun and pumping the supernatant into a waste liquid cylinder for the centrifugal tube provided with the Janus platinum nano motor, adding 600uL of MES solution into the centrifugal tube after the supernatant is completely taken away, and carrying out ultrasound by using a cell disruption ultrasonic machine; after the ultrasonic treatment is finished, adding 400uL of MES solution mixed with EDC and NHS prepared in the second step, fixing the centrifugal tube in a rotary mixer, and activating for 1-2 hours at a set rotating speed of 15;
fourthly, centrifuging by using a centrifugal machine after activation is finished, wherein the rotating speed of the centrifugal machine is 12000rpm, the working time is 6min, and the working temperature is 4 ℃; taking out the centrifugal tube after the centrifugation is finished, sucking the supernatant in the centrifugal tube by using a pipette gun and pumping the supernatant into a waste liquid cylinder for the centrifugal tube provided with the Janus platinum nano motor, adding 1mL of PBS solution into the centrifugal tube after the supernatant is completely taken away, and carrying out ultrasound by using a cell disruption ultrasonic machine;
fifthly, adding 0.1-1 mg of antibody for coating into the Janus platinum nano motor solution by using a pipette gun, and then fixing the centrifuge tube on a rotating disc to rotate for labeling for 4-6 hours;
sixthly, centrifuging by using a centrifugal machine after marking is finished, taking out the centrifugal tube after centrifugation is finished, sucking supernatant in the centrifugal tube by using a liquid transfer gun to the centrifugal tube provided with the Janus platinum nano motor, pumping the supernatant into a waste liquid cylinder, adding a PBS (phosphate buffer solution) solution into the centrifugal tube after the supernatant is completely taken away, and performing ultrasound by using a cell disruption ultrasonic machine; the ultrasonic power is 180W, 2 seconds of ultrasonic pause are set as a group, 2 groups are carried out totally, the ultrasonic duration is 4 seconds in total, and the ultrasonic environment is ice bath;
a seventh step of adding 10% BSA solution to the Janus platinum nanomotor solution with a pipette gun, followed by blocking the centrifuge tube by rotating on a rotating disk for 1-2 hours;
step eight, centrifuging by using a centrifuge after sealing is finished, wherein the rotating speed of the centrifuge is 12000rpm, the working time is 6min, and the working temperature is 4 ℃; taking out the centrifugal tube after the centrifugation is finished, sucking the supernatant in the centrifugal tube by a pipette gun and pumping the supernatant into a waste liquid cylinder for a Janus platinum nano motor, adding 1mL of 1-percent BSA solution into the centrifugal tube after the supernatant is completely removed, and carrying out ultrasound by using a cell disruption ultrasonic machine; the ultrasonic power is 180W, 2 seconds of ultrasonic pause are set as a group, 2 groups are carried out totally, the ultrasonic time is 4 seconds in total, the ultrasonic environment is an ice bath, and the antibody coupling is completed after the ultrasonic is finished;
ninth, spraying gold on the glass fiber treated by the glass fiber treatment solution by the Janus platinum nano motor solution coupled with the antibody according to the spraying amount of 2.0 uL/cm; after the gold spraying is finished, placing the glass fiber in a drying oven at 37 ℃ for drying for 24 hours;
tenth step, sticking absorbent paper on the top end of the PVC bottom card, and sticking an NC film on the middle part of the PVC bottom card; diluting the antibody for scribing to 1mg/mL by using an antibody diluent, scribing a detection line on an NC membrane according to the spraying amount of 1uL/cm, diluting the secondary antibody to 1mg/mL by using the antibody diluent, and scribing a control line on the NC membrane according to the spraying amount of 1 uL/cm; after the scribing is finished, the test card is placed into a drying oven at 37 ℃ to be dried for 24 hours;
step eleven, after drying, sticking the glass fiber to the lowest end of the PVC bottom card, and cutting the PVC bottom card into test strips with the width of 4mm by using a high-speed cutting machine;
and a twelfth step of adding a hydrogen peroxide solution into the sample solution to enable the mass fraction of the hydrogen peroxide solution to be 1-10wt%, and dropwise adding 90uL of the hydrogen peroxide solution into the test strip for antigen detection.
3. The Janus platinum nanomotor-based immunochromatographic detection method of claim 2, wherein after the first step of ultrasonication is completed, 0.4mL of Janus platinum nanomotor solution is taken to enter a 1.5mL centrifuge tube, 0.6mL of MES solution is added into the centrifuge tube, and the mixture is pumped and uniformly mixed by using a pipette, so that the concentration of the configured Janus platinum nanomotor solution is 1-5mg/mL, and the volume of the solution is 1mL; and then centrifuging by using a centrifuge, setting the rotation speed of the centrifuge to 12000rpm, working for 6min and working at 4 ℃.
4. The Janus platinum nanomotor-based immunochromatographic assay method of claim 2, wherein in the second step, the weighed two drugs are poured into a 1.5mL empty centrifuge tube, and 400uL of MES solution is added thereto by using a pipette gun, and the drugs are pumped and mixed uniformly.
5. The Janus platinum nanomotor-based immunochromatographic detection method of claim 2, wherein the third step is performed at a power of 180W for 2 seconds, and is performed for 2 groups of 2 times of 2 second pause, and the duration of the ultrasonic treatment is 4 seconds in total.
6. The Janus platinum nanomotor-based immunochromatographic detection method of claim 2, wherein the fourth step is performed at an ultrasonic power of 180W for 2 seconds, and is performed for 2 groups of 2-second pauses, and the ultrasonic duration is 4 seconds in total.
7. The Janus platinum nanomotor-based immunochromatographic assay method of claim 2, wherein in the fifth step, 0.25mg of the antibody for coating is added to the Janus platinum nanomotor solution by using a pipette gun, and then the centrifuge tube is fixed on a rotating disk and rotated for 4-6 hours for labeling.
8. The Janus platinum nano-motor-based immunochromatographic detection method of claim 2, wherein after the sixth step of marking is completed, the centrifugal machine is used for centrifugation, the rotation speed of the centrifugal machine is set to 12000rpm, the working time is 6min, the working temperature is 4 ℃, the centrifugal tube is taken out after the centrifugation is completed, for the centrifugal tube provided with the Janus platinum nano-motor, a liquid transfer gun is used for sucking the supernatant in the centrifugal tube and pumping the supernatant into a waste liquid tank, and after the supernatant is completely taken away, 1mL of PBS solution is added into the centrifugal tube.
9. The Janus platinum nanomotor-based immunochromatographic detection method of claim 2, wherein said seventh step is to add 100uL of 10% BSA solution to the Janus platinum nanomotor solution with a pipette gun, followed by fixing the centrifuge tube on a rotating disk and rotating for 1 hour for blocking.
10. A method for improving the antigen detection ability of an immunochromatographic test strip, which comprises the immunochromatographic detection method based on a Janus platinum nanomotor according to any one of claims 1 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210576462.2A CN115248310B (en) | 2022-05-25 | 2022-05-25 | Immunochromatography detection method based on Janus platinum nanomotor and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210576462.2A CN115248310B (en) | 2022-05-25 | 2022-05-25 | Immunochromatography detection method based on Janus platinum nanomotor and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115248310A true CN115248310A (en) | 2022-10-28 |
| CN115248310B CN115248310B (en) | 2023-08-15 |
Family
ID=83698027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210576462.2A Active CN115248310B (en) | 2022-05-25 | 2022-05-25 | Immunochromatography detection method based on Janus platinum nanomotor and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115248310B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120288852A1 (en) * | 2010-01-15 | 2012-11-15 | Richard Willson | Force Mediated Assays |
| US20140045179A1 (en) * | 2011-01-31 | 2014-02-13 | Joseph Wang | Nano/microscale vehicles for capture and isolation of target biomolecules and living organisms |
| CN104655836A (en) * | 2013-11-25 | 2015-05-27 | 国家纳米科学中心 | Immunochromatographic test strip, detection method by using immunochromatographic test strip, and application of immunochromatographic test strip |
| CN113056674A (en) * | 2018-09-25 | 2021-06-29 | 悉尼科技大学 | Quantification of analytes |
-
2022
- 2022-05-25 CN CN202210576462.2A patent/CN115248310B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120288852A1 (en) * | 2010-01-15 | 2012-11-15 | Richard Willson | Force Mediated Assays |
| US20140045179A1 (en) * | 2011-01-31 | 2014-02-13 | Joseph Wang | Nano/microscale vehicles for capture and isolation of target biomolecules and living organisms |
| CN104655836A (en) * | 2013-11-25 | 2015-05-27 | 国家纳米科学中心 | Immunochromatographic test strip, detection method by using immunochromatographic test strip, and application of immunochromatographic test strip |
| CN113056674A (en) * | 2018-09-25 | 2021-06-29 | 悉尼科技大学 | Quantification of analytes |
Non-Patent Citations (6)
| Title |
|---|
| ENHUI MA等: "An immunoassay based on nanomotor‐assisted electrochemical response for the detection of immunoglobulin" * |
| LIHONG SU等: "Competitive Lateral Flow Immunoassay Relying on Au–SiO2 Janus Nanoparticles with an Asymmetric Structure and Function for Furazolidone Residue Monitoring" * |
| XINLI LIU等: "Gas-propelled biosensors for quantitative analysis" * |
| YANG LI等: "Simultaneous Detection of Inflammatory Biomarkers by SERS Nanotag-Based Lateral Flow Assay with Portable Cloud Raman Spectrometer" * |
| 苏丽红: "纳米信号探针用于呋喃唑酮免疫层析检测方法的研究" * |
| 郑旭;崔海航;李战华;: "Janus球形微马达的自驱动机理研究:自扩散泳动与微气泡推进" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115248310B (en) | 2023-08-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Siebert et al. | Liposome immunomigration field assay device for Alachlor determination | |
| JPH0222560A (en) | Reactor for performing sequential analytic test | |
| JPH0346566A (en) | Reaction cassette for executing successive analytical test by non-centrifugal and non-capillary operation | |
| JPS6071957A (en) | Method of promoting immune reaction by ultrasonic treatment | |
| JP4972295B2 (en) | Immunoassay method and biochip | |
| Bowen et al. | Immunoassay interference by a commonly used blood collection tube additive, the organosilicone surfactant silwet L-720 | |
| JP2002511926A (en) | Assays using magnetic particles | |
| JPH0545361A (en) | Analysis for liquid sample and liquid sample analyzing substrate used for the analysis | |
| JPS6365369A (en) | Method for measuring antigen-antibody reaction | |
| AU2005303881A1 (en) | Device for carrying out an individual immunoassay in a fully automatic manner | |
| CN115248310A (en) | Immunochromatography detection method based on Janus platinum nanomotor and application | |
| CN210155150U (en) | Full-automatic magnetic particle chemiluminescence immunoassay analyzer | |
| WO2012127122A1 (en) | Method for performing a rapid test | |
| CN114167052A (en) | Kit for quantitative detection of NT-proBNP/ST2 by time-resolved fluorescence immunochromatography and application thereof | |
| US7184894B2 (en) | Quantitative measurement method and quantitative measurement chip for target substance | |
| JPS6327760A (en) | Analytic element with water-soluble polymer | |
| JP3598334B2 (en) | Measuring reagent and measuring method using quartz oscillator | |
| CN112462069B (en) | Fluorescence immunochromatography kit for detecting canine pancreatitis and preparation method thereof | |
| CN110726848A (en) | Thyroid stimulating hormone detection immune test paper and preparation method thereof | |
| CN117310186A (en) | Kit and method for detecting insulin-like growth factor binding protein-3 (IGFBP-3) | |
| CN116660538A (en) | Kit and detection method | |
| CN116165384A (en) | Kit for measuring cytokeratin 19 fragment by fluorescent immunochromatography and preparation method thereof | |
| CN116413446A (en) | Detection card and kit for detecting 25-OH VD content by one-step method | |
| CN115932289A (en) | 25-OH-VD detection kit and application thereof | |
| CN114578067A (en) | Multiple kit for detecting sex hormone by fluorescence immunoassay and preparation method thereof |
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 | ||
| CB03 | Change of inventor or designer information |
Inventor after: Ma Xing Inventor after: Guo Jinhong Inventor after: Guo Jiuchuan Inventor after: Li Yang Inventor before: Guo Jiuchuan Inventor before: Li Yang Inventor before: Guo Jinhong Inventor before: Ma Xing |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |