CN115436622A - Detection method of monomolecular protein, kit and application thereof - Google Patents
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Abstract
The invention discloses a detection method of single molecule protein and a kit and application thereof, relating to the field of biological detection.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a detection method of a single-molecule protein, a kit and application thereof.
Background
The immunoassay plays an important role in the field of detection of various diseases, the single-molecule immunoassay cannot be realized by the existing chemical light-emitting technology, and the single-molecule immunoassay technology is an ultrasensitive protein detection technology developed on the basis of the microfluidic technology.
Microfluidic technology (Microfluidics) is a technology for accurately controlling fluid in micro-nano scale space. It can integrate the basic operation units of sample preparation, reaction, separation, detection and the like in the chemical, biological and medical analysis process on a micron-scale chip. Compared with the traditional experimental method, the micro-fluidic chip has the advantages of miniaturization, integration, automation, sensitive reaction, less sample consumption and the like. In 2010, david Walt et al proposed a concept of "Digital ELISA" using a microfluidic chip technology, performed enzyme-linked labeling by an immunolabeling method using an antibody to capture and recognize an antigen, and developed a SimoA single-molecule immunodetection technology by detecting single-molecule-level protein molecules through a single-molecule enzymatic reaction, wherein the specific operation flow is shown in FIG. 1.
The detection of the monomolecular protein is realized by the combination of the SiMoA and the microporous array chip through the enzyme-linked immunosorbent assay of the magnetic beads. The operation process is as follows: (1) Capturing the antigen in the sample by using the magnetic beads with the surfaces marked with the capture antibodies; (2) Labeling the captured antigen with a Biotin (Biotin) -labeled detection antibody; (3) Adding streptavidin-galactosidase compound to combine with Biotin on the detection antibody; (4) Mixing the magnetic beads after reaction and cleaning with a substrate, loading the mixture into a detection chip containing a micropore array, enabling the magnetic beads to fall into micropores (about 3 mu m) completely matched with the size of the magnetic beads by using a magnetic field, and adding an oil phase to physically isolate the micropores; (5) The galactosidase-containing microwell produces a fluorescent product as a result of the catalytic substrate of the enzyme molecule; (6) And carrying out fluorescence imaging on the micropore array, and comparing the number of micropores emitting fluorescence signals with a standard curve to realize quantitative detection.
The SiMoA system actually adopts a single-molecule signal amplification method in digital PCR, antigen molecule enzyme-linked labeling is carried out through enzyme-linked immunity, and then a method of enzyme-catalyzed fluorescence substrate replaces PCR in the digital PCR technology to realize signal amplification. According to data published by the prior Quanterix official, the SiMoA system is an immunodetection system with the highest detection sensitivity in the current global range, the lower limit of cTnI detection reaches 0.05ng/mL, and the SiMoA system is the only official publication and can have a protein detection and nucleic acid molecule detection general technical platform. The detection sensitivity of the SimoA single-molecule immunodetection technology exceeds that of the traditional detection method by nearly 20000 times, however, the SiMoA system has some obvious defects: (1) the detection process is too complex; (2) The cost is high, and a micropore array with high processing precision is required; and (3) the detection takes long time, and usually needs more than 1 hour.
SMC systems are device-dependent single-molecule detection technologies. Different from the idea that SiMoA concentrates fluorescent products by reducing the volume of reaction units, SMC adopts a more direct form of laser focusing to improve the photon yield of a few fluorescent dye molecules of immune markers so as to realize the counting detection of single molecules. The operation flow is shown in fig. 2.
The specific steps of the SMC performing single-molecule immunodetection are as follows: capturing antigen by using an enzyme label plate or magnetic beads marked with capture antibody (1), carrying out fluorescence labeling on the antigen by using detection antibody marked with a plurality of fluorescent dye molecules (3), eluting the antigen and the detection antibody marked with fluorescence by using special eluent, transferring the eluted antigen and the detection antibody marked with fluorescence into a detection hole (4), and carrying out single molecule counting by using equipment. Currently, there are two generations of detection devices in SMC system, as shown in fig. 3, the first generation of detection device in SMC system is Erenna, which is a mobile fluorescent molecule scanning system. When scanning fluorescent molecules, it is necessary to pass the eluted fluorescent molecules in the solution through a very small detection window one by one. In the detection window, the high-power laser is focused to the micrometer scale by utilizing the laser focusing technology, so that the problem of signal superposition of a plurality of antigen-labeled fluorescent molecules can be avoided, the energy of an excitation light source of the detection window can be improved as much as possible under the optical limit scale, the photon yield of only a few fluorescent molecules of a single antigen label is improved, and the single-molecule signal is ensured to be distinguished from background noise. However, this detection method brings about several problems: (1) Because the detection window is very small, it takes a lot of time (tens of minutes) for the eluent with the volume of tens of microliters or even hundreds of microliters to completely flow through the detection window, and because of the interference of the operating environment, the liquid channel of the detection window is very easy to be blocked, and the micrometer-scale optical focusing of the elution solution (2) which needs to be specially filtered and processed is close to the limit of optical diffraction, so that the equipment has extremely strict requirements on the adjustment accuracy of an optical system and the stability of the operating environment of the equipment. To solve this problem, the second generation SMC device SMCxPRO was introduced by Merck. The second generation SMC system is compatible with the capture of magnetic beads and multi-well plates, but in order to reduce the background of the reaction and avoid the problem of light source or emitted light blocking due to the limited transparency of the magnetic beads, it is still necessary to elute the fluorescent molecules and use a dedicated detection well to load the detection solution for detection. The SMCxPRO changes the form of the flow-type scanning fluorescent molecules, and changes the mode of scanning three-dimensional eluent in the detection hole by rotating the light source, thereby obviously improving the efficiency of molecular counting. This detection method has been separated strictly from the concept of absolute counting of single molecules. Due to the fact that the fluorescent molecules in the solution have Brownian motion and the molecular motion tracks are unpredictable, the possibility of repeated counting or missing detection of the molecules exists in the detection process, the detection sensitivity is reduced to a certain extent, and algorithm-assisted correction is theoretically needed. Compared with the generation devices, the generation SMC system can improve the generation system significantly in the time consumption of a single complete test, but the single test still needs more than 4 hours. In summary, SMCxPRO can still only be used for scientific research level detection and analysis (actually, the positioning of the SMC technology by Merck corporation is limited to the scientific research field), and although the detection sensitivity of the system far exceeds the current mainstream of each large chemiluminescence technology platform, the characteristics in other aspects far do not meet the requirements of medical diagnosis products.
At present, a single-molecule immunoassay method based on an up-conversion fluorescent probe is also provided, and the method comprises the following specific operation procedures: (1) modifying a capture antibody on a detection substrate in advance; (2) Diluting a sample to be detected, then dropwise adding the diluted sample to a detection substrate, and combining the detected sample with a pre-modified capture antibody; (3) After the reaction is finished, washing the detection substrate, and dropwise adding an immunofluorescence probe (an up-conversion fluorescent nano material modified with a detection antibody); (4) And washing the detection substrate, placing the processed detection substrate under a fluorescence microscope, and counting the number of the immunofluorescence probes, wherein the obtained number is the number of the detected objects. According to the method, the high-concentration rare earth-doped up-conversion luminescent nanoparticles are used as the basis of the immunofluorescence probe, the result can be read by naked eyes under a fluorescence microscope, and quantification is carried out through the number of the immunofluorescence probe, so that the method is suitable for pathogens, microorganisms, macromolecular antigens or antibodies, and is wide in detectable range. However, the open reaction and post-treatment are difficult to avoid contamination of the sample to be tested, resulting in false positives. Meanwhile, the scheme requires high cost and complicated and long probe synthesis steps, and is not beneficial to popularization and promotion of the application.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a detection method of a monomolecular protein, a kit and application thereof.
The invention is realized by the following steps:
in a first aspect, the embodiments of the present invention provide the use of a reagent combination in the preparation of a detection kit for a single-molecule protein, the reagent combination comprising: the detection kit comprises a marker for generating a detection signal, a capture antibody capable of being combined with an object to be detected and a detection antibody capable of being combined with the object to be detected, wherein the capture antibody is fixed on the surface of a solid phase carrier, a first photocrosslinking agent is modified on the capture antibody, a second photocrosslinking agent and biotin are modified on the detection antibody, and streptavidin is marked on the marker.
In a second aspect, the embodiments of the present invention provide a kit for detecting a single-molecule protein, which includes the composition described in the previous embodiments.
In a third aspect, the embodiments of the present invention provide a method for detecting a single-molecule protein, which includes detecting a sample to be detected by using the single-molecule protein detection kit as described in the previous embodiments.
The invention has the following beneficial effects:
1. the detection time is short: the contact probability of the target protein and the capture antibody is increased through the high specific surface area of the magnetic bead carrier, the time required by the conventional ELISA reaction is reduced, the time required by antigen-antibody combination is reduced, the reaction time is greatly shortened, and the detection can be completed within about 30 min.
2. The detection sensitivity is high: through covalent crosslinking of a photocrosslinking agent, the binding force of ELISA reaction is increased, the change of the whole optical characteristics of the measured solution is converted into the recognition of single high-fluorescence particles, and the detection sensitivity is improved to a single molecular level.
3. The cost is low: does not need to use precision machining equipment, and has the advantages of easy integration, less reagent consumables, low cost and the like.
4. The requirement on detection equipment is low: the nano-scale fluorescent microspheres are used for converting the single-molecule signals into the single-fluorescent-microsphere signals to realize signal amplification, so that the requirement of detection equipment on an optical system is lowered.
5. The application range is wide: the kit is suitable for most immunodetection systems, and has wide application potential in the fields of cell biology, immunology, clinical medicine and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a diagram of the SiMoA technology principle (left), siMoA HD-1Analyzer apparatus (right);
FIG. 2 is a flow chart illustrating SMC technology operation;
FIG. 3 shows a first generation SMC detection device Erenna and a second generation SMCCxPRO;
FIG. 4 is a flow chart of the single molecule immunoassay provided herein;
FIG. 5 is a schematic diagram of a modified photocrosslinker on an antibody;
FIG. 6 is a graph showing the effect of illumination time on the test results;
FIG. 7 shows the detection sensitivity of p-Tau 217;
FIG. 8 shows the detection sensitivity of IL-10;
FIG. 9 shows the specificity of detection of IL-10.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
According to the invention, a traditional double-antibody sandwich structure is converted into a high-stability covalent compound through reactions such as photo-crosslinking, and a single-molecule double-antibody sandwich structure is converted into a single high-fluorescence particle signal by utilizing a high-affinity reaction between streptavidin-labeled high-fluorescence particles and a biotin-labeled double-antibody sandwich structure, so that a novel high-stability single-molecule immunoreaction system without enzyme amplification fluorescence signals is established; the whole optical characteristics of the traditional measuring solution are converted into the identification of single high-fluorescence particles, and the single-molecule-level immune quantitative detection is realized by scanning the signal of the single high-fluorescence particles.
Compared with the traditional immunodetection method for realizing quantitative detection by measuring the integral optical characteristics of the solution, such as chemiluminescence and enzyme-linked immunosorbent assay, the unimolecular immunodetection system constructed by the scheme realizes the breakthrough of the principle because the amplification of a fluorogenic substrate signal by an enzyme molecule is not required at the detection principle level, so that the sensitivity and the accuracy of the system are far higher than those of the traditional technology, the limitation of the sensitivity of the traditional detection method is overcome, and the high-sensitivity detection of low-abundance protein in a clinical sample is realized. Based on the Alzheimer disease blood marker, the method provides a necessary novel research method for the research and development of novel biomarkers and the research of deeper single cell level and single molecule level in the future.
Specifically, the embodiment of the present invention provides an application of a reagent combination in preparing a detection kit for a single-molecule protein, wherein the reagent combination comprises: the detection kit comprises a marker for generating a detection signal, a capture antibody capable of being combined with an object to be detected and a detection antibody capable of being combined with the object to be detected, wherein the capture antibody is fixed on the surface of a solid phase carrier, a first photocrosslinking agent is modified on the capture antibody, a second photocrosslinking agent and biotin are modified on the detection antibody, and streptavidin is marked on the marker.
In some embodiments, the molar ratio of the first photocrosslinker to the capture antibody is (5-1000): 1; the molar ratio of the second photocrosslinking agent to the detection antibody is (5-1000): 1. specifically, the molar ratio of the first photocrosslinker or the second crosslinker to the antibody is 5:1, 10: 1. 100. This molar ratio can be understood as: when the antibody modifies the photocrosslinking agent, the molar ratio of the photocrosslinking agent to the antibody is adopted; or the molar ratio of photocrosslinker to antibody after modification.
In some embodiments, the step of modifying the first photocrosslinker on the capture antibody or modifying the second photocrosslinker on the detection antibody comprises: and mixing the photocrosslinking agent and the antibody according to the molar ratio.
In some embodiments, the conditions of the mixing are: reacting at room temperature (5-25 deg.C) for 25-35 min, wherein the temperature can be 5 deg.C, 6 deg.C, 8 deg.C, 10 deg.C, 12 deg.C, 14 deg.C, 16 deg.C, 18 deg.C, 20 deg.C, 22 deg.C, 24 deg.C and 25 deg.C or any two ranges; the time may be any one of 25min, 26min, 28min, 30min, 32min, 34min and 35min or a range between any two.
In some embodiments, the conditions of the mixing are: reacting for 1.5-2.5 h at 0-4 ℃. Specifically, the temperature can be in the range of any one or two of 0 ℃, 1 ℃, 2 ℃,3 ℃ and 4 ℃; the time may be any one or a range between any two of 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2.0h, 2.1h, 2.2h, 2.3h, 2.4h and 2.5h.
The first photocrosslinking agent and the second photocrosslinking agent are both reagents containing photosensitive groups. In some embodiments, the first and second photocrosslinkers are independently selected from: any one or a mixture of at least two of the compounds containing one or at least two bisaziridine functional groups.
Specifically, the compound is selected from at least one of the following compounds 1 to 4.
In some embodiments, the solid support is selected from at least one of a magnetic bead, a plate, and a membrane; preferably, the solid phase carrier is a magnetic bead having a particle size of 1-100 μm, and specifically may be any one or a range between any two of 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm and 100 μm.
In some embodiments, the label is selected from at least one of a fluorescent dye and a nanoparticle-based label.
In some embodiments, the nanoparticle-based labels have a particle size of 20 to 5000nm. The particle diameter may be specifically in the range of any one or two of 20nm, 50nm, 100nm, 200nm, 400nm, 600nm, 800nm, 1000nm, 1200nm, 1400nm, 1600nm, 1800nm, 2000nm, 2200nm, 2400nm, 2600nm, 2800nm, 3000nm, 3200nm, 3400nm, 3600nm, 3800nm, 4000nm, 4200nm, 4400nm, 4600nm, 4800nm, and 5000nm.
In some embodiments, the fluorescent dye is selected from at least one of a fluorescein-based dye, a rhodamine-based dye, a Cy-series dye, an Alexa-series dye, and a protein-based dye.
In some embodiments, the nanoparticle-based labels include nanoparticles selected from at least one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles, and/or colloids selected from at least one of latex, colloidal selenium, colloidal metal, disperse dye, and dye-labeled microspheres.
In some embodiments, the colloidal metal is selected from at least one of colloidal gold and colloidal silver.
In some embodiments, the composition further comprises a detergent; preferably, the detergent comprises PBS.
The kit or the constructed single-molecule protein detection system provided by the invention has no specific limitation on the protein, and the protein with any molecular weight is detected at the molecular level based on the detection method constructed in the invention. In some embodiments, the single molecule protein detection kit can be a biomarker detection kit for a disease.
It is understood that the technical effects of high specificity and high sensitivity of the detection method of the single-molecule protein described herein are mainly due to the advantages of the detection system itself, rather than relying on antibodies. In some embodiments, the antibody can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or an antigen-binding fragment. Any antibody that can bind to the analyte is suitable for use in the method of the present invention. The antigen-binding fragment may be: VHH, F (ab') 2 Preferably, the antibody is at least one of a monoclonal antibody, a polyclonal antibody, and a recombinant antibody.
The embodiment of the invention provides a detection kit of a single-molecule protein, which comprises the composition as described in any embodiment of the invention.
In addition, the embodiment of the invention provides a method for detecting a monomolecular protein, which comprises the step of detecting a sample to be detected by using the kit for detecting a monomolecular protein as described in any of the preceding embodiments.
In some embodiments, the detecting comprises the steps of: mixing a sample to be detected with the capture antibody and the detection antibody in the kit for reaction, and irradiating the mixed reaction product under the action of ultraviolet light to enable the first photocrosslinking agent and the second photocrosslinking agent to generate crosslinking reaction; adding a marker into the product after the crosslinking reaction, washing after mixing to remove the free marker in the product, and detecting the signal of the marker.
In some embodiments, the wavelength of the ultraviolet light is 300 to 450nm; specifically, the wavelength may be in a range of any one or two of 300nm, 320nm, 340nm, 360nm, 380nm, 400nm, 420nm, 440nm, and 450 nm.
In some embodiments, the time of the irradiation is greater than or equal to 0.5min; preferably 0.5-16 min, specifically any one or a range between any two of 0.5min, 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, 12.5min, 13min, 13.5min, 14min, 14.5min, 15min, 15.5min and 16 min.
In some embodiments, the instrument employed for the detection comprises a fluorescence microscope.
In some embodiments, the detection method is not directed toward the diagnosis or treatment of a disease.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
A method for detecting single molecule protein, the reaction principle is shown in figure 4, which comprises the following steps.
(1) Firstly, modifying photo-crosslinking groups Diazirine (bis-aziridine) on a capture antibody and a detection antibody respectively, and the principle is shown in FIG. 5;
the process is as follows:
dissolving the protein (antibody) in PBS at a concentration of 10mg/mL;
2mg of cross-linking agent NHS-ester diazirines are dissolved in 888 microliter DMSO to prepare 10mM mother solution;
adding cross-linking agent NHS-ester diazirines into the protein solution to ensure that the final concentrations of the cross-linking agent NHS-ester diazirines and the protein are 1mM and 20 mu M respectively;
reacting at room temperature for 30 minutes or at 4 ℃ for 2 hours;
adding a quenching reagent Tris to quench the reaction, wherein the final concentration is 100mM, and reacting for 5 minutes at room temperature or 15 minutes at 4 ℃;
the unreacted cross-linking agent NHS-ester diazirines was removed by dialysis with dialysis bag and the protein was lyophilized to make 100. Mu.M solution for use.
(2) Modifying the capture antibody on the surface of a carboxyl magnetic bead (Suzhou-degree biotechnology, co., ltd., product number CMP1003 CA) through EDC/NHS esterification reaction;
(3) After that, the modified magnetic beads in the step (2) were washed for a plurality of times (PBS washing 3 times) to remove the excess capture antibody, thereby obtaining MB @ Capture Ab (magnetic beads modified with capture antibody);
(4) Mixing 10 μ L of MB @ Capture Ab (10 mg/mL magnetic bead) in step (3) with 1 μ L of target protein (different concentrations: 1fg/mL, 10fg/mL, 100fg/mL, 1pg/mL, 10pg/mL, 100pg/mL, 1ng/mL, 10ng/mL, 100 ng/mL), 10 μ L of Biotin (Biotin) -modified detection antibody (10 ng/mL), and 29 μ L of PBS buffer solution, placing on a vortex shaker for reaction for 5 minutes;
(5) After the reaction is finished, washing the reaction system for 3 times by using PBS (phosphate buffer solution) to remove redundant detection antibodies to obtain a solution I;
(6) Placing the solution I under ultraviolet light (with the wavelength of 395 nm), and irradiating for a proper time (3 min) to initiate a crosslinking reaction between Diazirine and Diazirine on adjacent antibody molecules to obtain a solution II;
(7) Adding Streptavidin (SA) -modified fluorescent nanoparticles (FG 0400SA, particle size 400 nm) into the solution II, and washing with 1 × PBS to remove excessive fluorescent nanoparticles after the reaction is completed (1 min) to obtain solution III;
(8) After washing, the solution III is assisted by a fluorescence microscope to count the fluorescence signals of the magnetic beads (count fluorescence points on a unit area), count and analyze;
(9) And (5) processing the fluorescence signal data obtained in the step (8) and performing single-molecule-level accurate analysis on the target protein to be detected.
Test example 1
Influence of illumination time on detection results.
Based on the method provided by embodiment 1, multiple groups of experimental groups are set based on different ultraviolet light irradiation times, and the influence of the irradiation times on the detection result is verified. In the test example, the substance to be tested is p-Tau217 for exemplary verification, the capture antibody is a p-Tau217 monoclonal antibody, and the detection antibody is biotin-modified goat anti-rabbit IgG. The experimental result shows that the signal is gradually increased along with the increase of the irradiation time within 0-3min, and the signal is kept unchanged within 3-16min, which indicates that the signal is saturated, so that the optimal illumination time is 3min.
The results are shown in Table 1 and FIG. 6.
TABLE 1 test results
Further, the detection sensitivity of p-Tau217 was verified, and the results are shown in Table 2 and FIG. 7. The detection result shows that the method has high detection sensitivity, the lower detection limit can be up to 0.8fM, and the method is suitable for scientific research and clinical detection and meets the differentiation requirements of different clients on different detection occasions.
TABLE 2 test results
Test example 2
Sensitivity of detection of IL-10.
Based on the method provided by embodiment 1, the sample to be tested is IL-10 for exemplary verification, wherein the adopted capture antibody is interleukin 10 monoclonal antibody, and the detection antibody is biotin-modified goat anti-rabbit IgG. The experimental result shows that the more fluorescent nanoparticles are detected with the increase of the concentration, the larger the signal value is, and the sensitivity is high, and the lower detection limit is as low as 0.1fM.
The results are shown in Table 3 and FIG. 7.
TABLE 3 test results
Remarking: the fluorescent particle number is the number of fluorescent particles.
The specificity of detection is verified, the target concentration is 10fM, and the detection results are shown in Table 4 and FIG. 9. There was a strong signal response to IL-10 targeting 10fM, whereas the 10fM non-targets IL-4, IL-1 β, PSA, TNF- α were close to the signal of the blank, with essentially no signal response.
TABLE 4 test results
Test example 3
The influence of the method provided by the invention on the detection limit is verified.
Based on the method provided in example 1, the samples to be tested are IL-10 and IL-1 beta for exemplary verification, and compared with the method of Quanterix (reference result, JACS 2020 Ultrasensive Detection of Attomolar Protein Concentrations by medicine Single Molecule Assays). The method comprises the following specific steps:
a) In each assay, 10 μ L of antibody-coated beads (100000 particles in total) and 10 μ L of biotinylated detection antibody were added to 100 μ L of protein sample, and the plate was then sealed and shaken for 1h to form an immune complex. The plate washer was washed 6 times with system wash buffer 1 (quantrix) in a BioTek405TS microplate washer, then resuspended in 100. Mu.L of streptavidin-DNA conjugate and the sample diluted with 5mM EDTA. The plate was shaken for 15min for streptavidin-DNA labeling of the immune complexes and washed 8 times with system wash buffer 1 in a microplate washer. After washing, the beads were transferred to a new 96-well plate, washed once more with 200. Mu.L of system wash buffer 1, and suspended in 60. Mu.L of RCA solution.
b) The RCA solution consisted of a 0.5mM mixture of deoxynucleotides (New England Biolabs),0.33U/ul phi29 DNA polymerase (Lucigen), 0.2mg/ml BA, 1nM ATTO 647N labeled DNA probe (integrated DNA technology) and 0.1% Tween-20, reaction buffer consisting of 50mM Tris-HCl (pH 7.5), 10mM (NH) 4 ) 2 SO 4 And 10mM MgCl 2 And (4) forming.
c) Dithiothreitol (DTT) was removed from phi29 polymerase solution provided by the manufacturer using a Zeba spin desalting column (7K MWCO, thermo Fisher Science). After shaking the plate at 37 ℃ for 1h, 150. Mu.L of PBS and 5mM EDTA were added to stop the RCA reaction. Washed 2 times with 200. Mu.L of drop-coating buffer (50 mM Tris-HCl,50mM NaC L,0.1% Tween-20,0.5% BSA), concentrated to 10-15. Mu.L, then resuspended and dropped onto microscope slides. The drop-coated beads were dried for 10-15min to form a monolayer film, which was then counted and quantified using a Quanterix apparatus.
The capture antibodies adopted by the experimental group and the control group are respectively interleukin 10 monoclonal antibody and interleukin 1 beta monoclonal antibody, and the detection antibodies are biotin-modified goat anti-rabbit IgG.
The results are shown in Table 5. The results show that the detection sensitivity of the method is slightly higher than that of the Quanterix method.
TABLE 5 test results
The detection times were compared as follows. The detection time of the method is far shorter than that of a Quanterix method.
TABLE 6 test results
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The application of a reagent combination in preparing a detection kit for single-molecule protein is characterized in that the reagent combination comprises: the detection kit comprises a marker for generating a detection signal, a capture antibody capable of being combined with an object to be detected and a detection antibody capable of being combined with the object to be detected, wherein the capture antibody is fixed on the surface of a solid phase carrier, a first photocrosslinking agent is modified on the capture antibody, a second photocrosslinking agent and biotin are modified on the detection antibody, and streptavidin is modified on the marker.
2. The use of claim 1, wherein the molar ratio of the first photocrosslinker to the capture antibody is (5-1000): 1; the molar ratio of the second photocrosslinking agent to the detection antibody is (5-1000): 1;
preferably, the first and second photocrosslinkers are independently selected from: any one of or a mixture of at least two of the compounds containing one or at least two diazirine functional groups.
3. The use of claim 1 or 2, wherein the solid support is selected from at least one of a magnetic bead, a plate, and a membrane;
preferably, the solid phase carrier is a magnetic bead;
preferably, the label is selected from at least one of a fluorescent dye and a nanoparticle-based label;
preferably, the particle size of the nanoparticle-based marker is 20 to 5000nm.
4. The use according to claim 3, wherein the fluorescent dye is selected from at least one of a fluorescein-based dye, a rhodamine-based dye, a Cy-based dye, an Alexa-based dye, and a protein-based dye;
preferably, the nanoparticle-based label includes nanoparticles selected from at least one of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles, and/or colloids selected from at least one of latex, colloidal selenium, colloidal metal, disperse dye, and dye-labeled microspheres;
preferably, the colloidal metal is selected from at least one of colloidal gold and colloidal silver.
5. The use according to claim 3, wherein the composition further comprises a detergent;
preferably, the detergent comprises PBS.
6. A kit for detecting a monomolecular protein, comprising the composition according to any one of claims 1 to 5.
7. A method for detecting a monomolecular protein, which comprises detecting a sample to be detected using the kit for detecting a monomolecular protein according to claim 6.
8. The method for detecting a single-molecule protein according to claim 7, wherein the detection comprises the steps of:
mixing a sample to be detected with the capture antibody and the detection antibody in the kit for reaction, and irradiating the mixed reaction product under the action of ultraviolet light to enable the first photocrosslinking agent and the second photocrosslinking agent to generate crosslinking reaction;
adding a marker into a product after the crosslinking reaction, washing after mixing to remove the free marker in the product, and detecting the signal of the marker.
9. The method for detecting a monomolecular protein according to claim 8, wherein the irradiation time is preferably 0.5min or more;
preferably, the irradiation time is 0.5-16 min;
preferably, the wavelength of the ultraviolet light is 300-450 nm;
preferably, the wavelength of the ultraviolet light is 350nm.
10. The method for detecting a single-molecule protein according to claim 8 or 9, wherein the detection is performed by using an apparatus comprising a fluorescence microscope;
preferably, the detection method is not directed towards the diagnosis or treatment of a disease.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002016635A2 (en) * | 2000-08-25 | 2002-02-28 | Friz Biochem Gmbh | Method for marking chemical substances |
| US20100183504A1 (en) * | 2007-06-14 | 2010-07-22 | Fanqing Frank Chen | Multimodal imaging probes for in vivo targeted and non-targeted imaging and therapeutics |
| CN102460174A (en) * | 2009-05-14 | 2012-05-16 | 牛津大学之校长及学者 | Clinical diagnosis of hepatic fibrosis using a novel panel of low abundant human plasma protein biomarkers |
| CN104487846A (en) * | 2012-06-22 | 2015-04-01 | 生物辐射实验室股份有限公司 | Human factor XIII as a normalization control for immunoassays |
| WO2015097313A1 (en) * | 2013-12-27 | 2015-07-02 | The University Of Hong Kong | Metal chelation-based fluorescent probes for protein or other biomolecule labeling in cells |
| CN108384786A (en) * | 2017-02-03 | 2018-08-10 | 天津医科大学 | A kind of protein-bonded detection method of aptamer |
| CN110763834A (en) * | 2018-07-25 | 2020-02-07 | 福建广生堂药业股份有限公司 | Method, reagent and kit for detecting content of immune marker |
| CN111004329A (en) * | 2019-12-06 | 2020-04-14 | 南京师范大学 | Protein molecule weak interaction capture magnetic bead based on light affinity covalent connection and preparation method and application thereof |
| CN111771126A (en) * | 2019-01-30 | 2020-10-13 | 苏州宇测生物科技有限公司 | Single-molecule quantitative detection method and detection system |
| CN112229774A (en) * | 2020-12-17 | 2021-01-15 | 生物岛实验室 | Method and device for detecting molecules |
-
2022
- 2022-09-26 CN CN202211176564.1A patent/CN115436622B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002016635A2 (en) * | 2000-08-25 | 2002-02-28 | Friz Biochem Gmbh | Method for marking chemical substances |
| US20100183504A1 (en) * | 2007-06-14 | 2010-07-22 | Fanqing Frank Chen | Multimodal imaging probes for in vivo targeted and non-targeted imaging and therapeutics |
| CN102460174A (en) * | 2009-05-14 | 2012-05-16 | 牛津大学之校长及学者 | Clinical diagnosis of hepatic fibrosis using a novel panel of low abundant human plasma protein biomarkers |
| CN104487846A (en) * | 2012-06-22 | 2015-04-01 | 生物辐射实验室股份有限公司 | Human factor XIII as a normalization control for immunoassays |
| WO2015097313A1 (en) * | 2013-12-27 | 2015-07-02 | The University Of Hong Kong | Metal chelation-based fluorescent probes for protein or other biomolecule labeling in cells |
| CN108384786A (en) * | 2017-02-03 | 2018-08-10 | 天津医科大学 | A kind of protein-bonded detection method of aptamer |
| CN110763834A (en) * | 2018-07-25 | 2020-02-07 | 福建广生堂药业股份有限公司 | Method, reagent and kit for detecting content of immune marker |
| CN111771126A (en) * | 2019-01-30 | 2020-10-13 | 苏州宇测生物科技有限公司 | Single-molecule quantitative detection method and detection system |
| CN111004329A (en) * | 2019-12-06 | 2020-04-14 | 南京师范大学 | Protein molecule weak interaction capture magnetic bead based on light affinity covalent connection and preparation method and application thereof |
| CN112229774A (en) * | 2020-12-17 | 2021-01-15 | 生物岛实验室 | Method and device for detecting molecules |
Non-Patent Citations (2)
| Title |
|---|
| CHEN-YO FAN等: "Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies", 《CHEMICAL SCIENCE》, vol. 10, no. 37, pages 8600 - 8609 * |
| 何亚峰等: "基于亲和探针的药物靶点鉴定技术研究进展", 《药学进展》, no. 01, pages 30 - 40 * |
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