Disclosure of Invention
The invention aims to solve the problems of low oxygen reduction electrocatalytic activity and low electrochemical stability of the silver-based nano catalyst at present.
Based on the above, the invention provides a multiplex immune molecule detection method, which comprises the following steps:
obtaining coded microspheres with capture molecules connected to the surfaces;
capturing a target immune molecule through the capture molecule, adding an enzyme-labeled reagent, and forming an enzyme-labeled immune sandwich complex on the surface of the coded microsphere;
driving coded microspheres with immune sandwich complexes modified on the surfaces into micropores of a microplate and sealing;
and (3) detecting the micro-pore plate by light excitation of the micro-pore plate after a preset time.
In some embodiments, the encoded microsphere includes a body of polymeric material and encoded material distributed in the body of polymeric material.
In some embodiments, the encoded microsphere further comprises magnetic nanoparticles orderly distributed in the body of polymeric material.
Preferably, the particle size of the encoded microsphere is 0.5-50 μm, and the particle size of the magnetic nanoparticle is 1-100 nm.
In some embodiments, the encoded microsphere comprises at least two luminescent encoding materials;
preferably, the luminescent coding material is an organic fluorescent material or an inorganic fluorescent material;
preferably, the luminescent coding material is at least one of organic dye and quantum dot.
In some embodiments, the method of preparing the encoded microsphere comprises the steps of:
mixing at least two luminescent materials and microspheres in a polymer material, dispersing the polymer solution in a water phase through a multi-coupling physical field to form uniform liquid drops, and then wrapping the luminescent materials and the magnetic nanoparticles in the liquid drops through a cross-linking polymerization reaction to obtain the coded microspheres.
In some embodiments, each of the encoded microsphere surfaces captures one target immune molecule.
In some embodiments, 1 encoded microsphere is contained in each of the microwells.
In some embodiments, encoded microspheres surface-modified with immune sandwich complexes are driven into the microwells of a microwell plate and sealed by an electric field.
The invention also provides a kit for detecting immune molecules, which comprises a coded microsphere, a detection molecule and a hybridization buffer solution, wherein the surface of the coded microsphere is connected with a capture molecule, the coded microsphere comprises a polymer material main body, magnetic nano particles and a coding material, and the magnetic nano particles and the coding material are distributed in the polymer main body material.
In some embodiments, the kit further comprises a microplate chip;
preferably, the volume of individual microwells on the microwell plate chip is (20-100) x 10-15L.
According to the invention, each detection molecule is digitized under the detection condition of the traditional analog signal ELISA, so that the protein detection reaches the femtocells (fg/ml), and the sensitivity is 1000 times higher than that of the traditional ELISA method. And the sample detection mode of the fluorescent coding liquid phase chip is adopted, at least 15 marker proteins can be detected, and the fluorescent coding liquid phase chip has extremely high application value in the aspects of saving sample size and improving detection research. The invention has great advantages especially for single molecule detection, and has wide application prospect in life science, in-vitro diagnosis, companion diagnosis, blood screening, drug research and development and the like.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It is to be noted that all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless defined otherwise. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention provides a method for giving up a macroscopic continuous signal by intensively detecting all magnetic particles of an immune sandwich compound with enzyme marks in detection fundamentally, and independently detecting each magnetic particle in a volume sealed in a femto-cell scale, and simultaneously detecting>10,000 magnetic particles. Under this volume the chemical signal generated by a single enzyme molecule can be detected. When the concentration of the molecules to be detected is in the fg/ml level, most of the magnetic particles are not marked by enzyme molecules and do not generate signals. The remaining magnetic particles are mostly labeled with one enzyme molecule, and a small fraction is labeled with two or more enzyme molecules, thereby generating a chemical signal that can be detected by the system. The poisson distribution (aeb= -ln (1-fon)) was followed between the ratio of the magnetic beads generating the chemical signal to the total magnetic beads (fon) and the ratio of the total number of marker enzyme molecules to the total number of magnetic beads (AEB, average Enzyme per Bead). When the concentration of the object to be detected reaches the pg/ml level, most of the magnetic particles after the sample treatment have 1-10 marker enzyme molecules (aeb=1-10), AEB still has positive relation with the concentration of the object to be detected, and aeb=ibead /Ienzyme ，Ibead Is the average value of signals generated by all magnetic beads in a single test, Ienzyme Is a single sheetAverage value of signal generated by individual enzyme molecules, Ienzyme ＝fon*Ibead AEB, at this time Ibead Mean the average signal value of all the beads generating signals in a single test, AEB= -ln (1-fon), calculated with the concentration of the analyte at the fg/ml level, fon<0.5 sample data, where the calculated AEB is more accurate and most of the signal-producing magnetic particles carry only one marker enzyme molecule, thus ensuring the calculated Ienzyme Accuracy of (c) in the specification). And (3) testing standard substances of the to-be-tested objects with different concentrations by using positive correlation of the concentrations of AEB and molecules of the to-be-tested objects, linearly fitting the AEB value and the concentration value of the to-be-tested object to draw a calibration curve, testing an unknown sample by using the same method, and taking the obtained signal value into the standard curve by using an interpolation method to obtain the concentration of the to-be-tested object in the unknown sample. By adopting a method of singly detecting each magnetic particle after being sealed, the detection sensitivity can be improved from the magnitude of pg/ml to the magnitude of fg/ml by about 1000 times.
In some preferred embodiments, multiple protein indicators are detected simultaneously for one assay. Different capture antibodies are coupled to different fluorescent coded magnetic particles, and the same sample is detected after mixing in an equal ratio.
Two or more fluorescent materials (organic and inorganic fluorescent molecules, including quantum dots and the like) are mixed with magnetic nano particles and other functional nano particles in a plurality of polymer materials (different molecular weights and different functional groups) which are required to be synthesized, a polymer solution is dispersed in a water phase through a multi-coupling physical field to form uniform liquid drops/micro-reaction groups, then the functional materials are wrapped and buried in the polymer microspheres through crosslinking and polymerization reaction, and reactive oligomeric polymers are exposed on the surfaces of the microspheres through micro-phase separation. The microspheres can be subjected to further surface chemical reaction through the exposed oligomeric macromolecules or other surface macromolecules of the microspheres, so that the surface achieves weak nonspecific affinity to nucleic acid molecules, the specific coupling reaction activity is strong, the density is high, the microspheres can be tolerated under different reaction conditions (organic phase and high temperature), and finally the controllable capture antibody for specifically identifying target molecules can be coupled on the surfaces of the microspheres.
For a particular analyte molecule, the capture antibody and the detection antibody each specifically recognize a different epitope with high affinity. Affinity and specificity are characterized and tested by surface plasmon resonance, protein chip and other methods. Antibodies with higher affinity are used as capture antibodies, and antibodies with relatively lower affinity are used as detection antibodies. The detection antibody needs to be labeled correspondingly according to enzyme, such as biotin, digoxin and the like. The surface of the magnetic particles is provided with functional groups such as epoxy, carboxyl and the like, and after the capture antibody is covalently fixed on the surface of the magnetic particles under the condition of a proper catalyst (EDC) and pH, the surface of the magnetic particles is required to be blocked by a proper blocking agent such as whey protein, bovine blood albumin and the like so as to reduce the nonspecific adsorption of the surface of the magnetic particles.
The enzyme-labeled immune sandwich complex can be formed by a three-step method, a two-step method or a one-step method. The three-step method is to add magnetic particles with capture antibodies to an unknown sample. After incubation at 25C with shaking for 30 minutes to 3 hours. Detection antibody was added and washed after incubation for 30 minutes to 3 hours with 25C shaking. The enzyme-labeled reagent was added and incubated at 25C with shaking for 30 minutes to 3 hours followed by washing. The two-step method is to add magnetic particles with capture antibodies and detection antibodies to the unknown sample and wash after incubation for 30 minutes to 3 hours with 25C shaking. The enzyme-labeled reagent was added and incubated at 25C with shaking for 30 minutes to 3 hours followed by washing. The one-step method is to add magnetic particles with capture antibodies, detection antibodies, and enzyme-labeled reagents to an unknown sample, and wash after incubation for 30 minutes to 3 hours with 25C shaking. The enzyme-labeled reagent needs to amplify chemical signals such as horseradish peroxidase, galactosidase and the like, and also needs to recognize detection antibodies such as biotin on streptavidin recognition detection antibodies, rabbit anti-mouse antibodies recognition detection antibodies of mouse origin and the like.
The composite structure of the microsphere-capture antibody-target molecule-detection antibody-marking enzyme is successfully formed, and the reaction of a solution is catalyzed by the marking enzyme molecule carried at the tail end in a reaction substrate, so that the reaction substrate molecules with multiple orders of magnitude are produced by utilizing the catalysis of one catalyst molecule, thereby realizing the effective amplification of signals.
Single molecule detection based on microsphere composite structure and microplate chip: because the traditional chemiluminescent catalysis mode is used, in order to achieve the single-molecule sensitivity without amplification, the concentration of the catalyst needs to be amplified to the greatest extent, so that the micro-reactor mode is adopted to place each microsphere composite structure in one micropore, the volume of the micropore is about 10e-15 liters, and then each micropore (oil phase or polymer film and the like) is sealed and locked, so that each microsphere composite structure is in a single small volume, the concentration of only one catalyst molecule on the composite structure is increased by a plurality of orders of magnitude, and the reflection of the single micropore is catalyzed sufficiently.
The density of microwells on a microwell plate chip determines the amount of data collected and the detection dynamic range. Closely spaced microwells need to be clearly resolved by optical detection and ensure that there is only one or no microspheres in each microwell. The disposable microplates were produced by injection molding. Non-disposable microwells can be fabricated by a chip MEMS method.
Assembling a microsphere composite structure and micropores: in order to ensure efficient and rapid pairing and assembly of the microspheres and the micropores one by one, the following methods can be adopted: 1. the gravity can naturally sink the microspheres into the micropores by utilizing the characteristic that the density of the microspheres is greater than that of water; 2. since the microspheres can encapsulate magnetic materials, magnetic force can be used to manipulate the microspheres into the microwells; 3. because the dielectric constant of the microsphere has a great difference with that of the solution, dielectrophoresis force can be generated by applying a non-uniform alternating electric field to push the microsphere into the micropores, and after the reaction detection is finished, the direction of the electric field frequency is adjusted to change the direction of the force so as to push the microsphere out of the micropores, thereby realizing the reutilization of the micropores.
If the detection of single molecules with extremely low concentration is required, the proportion of the microsphere to the target molecules needs to be adjusted to optimize according to the Poisson distribution, so that only one or no target molecules are grabbed on one microsphere.
Sample processing based on microspheres and detection equipment based on microplates
Sample treatment: the sample treatment part of the device integrates liquid transfer, mixing, magnetic attraction, liquid pre-storage, liquid path cleaning and other modules, and the sample is correspondingly diluted by an on-line dilution system integrated in the device and then mixed with reagents such as magnetic beads loaded with detection antibodies, enzyme-labeled reagents, washing liquid and the like for washing and the like. And finally obtaining the pure magnetic particle solution of the enzyme-labeled immune sandwich compound.
And (3) detection: the detection part of the device integrates modules such as multicolor fluorescence excitation, forward scattering imaging, fluorescence emission filtering, magnetic attraction and alternating electric field control, the microspheres with multiple fluorescence codes are manipulated into a micro-pore plate in the mode, the surface of the micro-pore plate is sealed by utilizing oil phase through liquid path automatic control, after catalytic reaction occurs, fluorescence with different wavelengths is used for near-field excitation, images under different filtering wavelengths are obtained by utilizing forward scattering light imaging photographing, and then the microspheres are manipulated out of the holes for liquid path cleaning.
Software and automatic control: all modules are automatically controlled through a bottom layer program, the software integrates an image automatic identification analysis module, the system automatically identifies the brightness in each micropore and obtains the distribution of the micropore brightness, whether reactions occur in each micropore or not is automatically judged, and the microsphere numbers corresponding to each reaction are automatically judged. The dynamic detection range of the analog signal is obtained through brightness analysis, and the number of the generated reactions of the microspheres with the same number is used as the dynamic detection range of the digital signal.
Further description will be provided below in connection with specific examples.
Example 1
High sensitivity detection for simultaneous detection of IL-2 and IL6 cytokines
1. Two kinds of fluorescent coding microsphere synthesis: styrene monomer, polymethyl methacrylate, initiator, crosslinking agent and acrylic acid oligomer are mixed in chloroform to prepare high polymer solution. Taking two 4.5mL high polymer solutions, namely a reaction solution 1 and a reaction solution 2, adding 0.5mL rhodamine, 0.5mL fluorescein and 90mg nano magnetic particles into the reaction solution 1, and adding 0.5mL rhodamine, 0.5mL fluorescein and 90mg nano magnetic particles into the reaction solution 2. And then placing the reaction solution 1 and the reaction solution 2 in two reactors with 300mL of deionized water and surfactant respectively, uniformly dispersing the reaction solution 1 and the reaction solution 2 into micro-emulsion droplets with the size of about 10 micrometers under the conditions of stirring, ultrasonic treatment and surface force adjustment, adding an initiator into the solution and heating to carry out polymerization crosslinking reaction, slowly dissolving chloroform in each droplet into water and volatilizing after 24 hours, polymerizing and crosslinking monomers, and finally forming the fluorescent coding microsphere 1 and the fluorescent coding microsphere 2. The surfaces of the two high molecular fluorescent coding microspheres are provided with carboxyl functional groups.
2. Microspheres 1 and 2 were coupled to IL-2 and IL-6 capture antibodies, respectively, and 1mg of microspheres were dispersed in 1ml of PBS buffer, 5mg of EDC and 5mg of Sulfo-NHS were added, mixed well and kept stirring for 10 minutes, and after washing with 1ml of PBS, 50ug of IL-2 capture antibody was added. Incubate for 30 minutes to 3 hours at room temperature with shaking. After adding a blocking agent such as BSA, the mixture was incubated at room temperature for 30 minutes with shaking, and the microspheres 1 were washed by magnetic separation and finally dispersed in PBS. Mixing the loaded magnetic beads 1 and 2 in equal volume, wherein the concentration of each magnetic bead is 0.5mg/ml
3. IL2 and IL6 standards were spiked to 10% bovine serum at concentrations of (0,0.001,0.005,0.01,0.3,1.0, and 10 pg/ml), respectively. 100ul of each standard was taken, 50ul of the medium magnetic bead mixed solution was added, and after incubation for 3 hours with 25C shaking, washed three times with 5 XPBS+0.1% Tween. Dispersing into 100ul PBS, adding 50ul 1ug/ml IL2 detection antibody and 50ul 1ug/ml IL6 detection antibody, incubating for half an hour at 25C shaking, and washing with 5 XPBS+0.1% Tween three times. Dispersing into 100ul PBS, and adding 50ul streptavidin-beta-galactosidase conjugated enzyme labeling reagent. Wash four times with 5x pbs+0.1% tween. Dispersed into 100ul of enzyme substrate solution. Samples containing unknown concentrations of the analyte are treated in the same manner.
4. Adding the microsphere composite structure into a reactor with a micropore plate chip through microfluid, adding 10MHz alternating current, pushing the microsphere into the micropore, sealing the surface of the micropore by silicone oil, reacting for 2 minutes, using 488nm wavelength light to excite, photographing an optical filter 1, photographing an optical filter 2, using 532 nm wavelength light to excite, photographing an optical filter 3, flowing in ethanol, flowing in cleaning liquid, changing the alternating current frequency to 10kHz, flowing in cleaning liquid, and cleaning the reactor.
5. Data processing and concentration determination. The image processing software first identified all the beads and classified the beads according to the intensity of fluorescence under filter 1 and filter 2 (bead 1, with IL-2 capture antibody and bead 2, with IL-6 capture antibody). Calculating the marker enzyme molecule for each concentration of each magnetic beadA ratio of the total number to the total number of magnetic beads (AEB, average Enzyme per Bead). When the concentration of the analyte is low, the ratio of the magnetic beads generating the chemical signal to the total magnetic beads (fon<0.5 Aeb= -ln (1-fon)). When fon is>0.5, aeb=ibead/Ienzyme, ibead is the average of all magnetic bead generated signals in a single test, ienzyme is the average of single enzyme molecule generated signals, Ienzyme Let fon×ibead/AEB, aeb= -ln (1-fon), calculated Ienzyme With all fons<Data for 0.5 samples. And after the AEB value and the concentration value of the object to be measured are subjected to linear fitting to draw a calibration curve, testing the unknown sample by using the same method, and taking the obtained AEB value into a standard curve by using an interpolation method to obtain the concentration of the object to be measured in the unknown sample. IL-2 concentration was 1.1pg/ml and IL-6 concentration was 0.8pg/ml as measured by the above method.
6. Determination of detection Limit
And (3) measuring the sample with the concentration of 0 for 10 times in parallel, and taking the standard deviation of the average value of the measured AEB value added with 3 times into a standard curve of 4. The solubility of the to-be-measured object obtained in the standard curve is the detection limit of the method. IL-2 detection limit was 0.069pg/ml, and IL-6 detection limit was 0.030pg/ml.
FIG. 1 shows the signals of the marker enzymes (galactosidase) detected by the forward scatter imaging module after oil sealing when fluorescent encoding microspheres modified with sandwich complexes were introduced into a microplate when IL-2 and IL-6 cytokine samples of unknown concentrations were measured in example 1. Each microwell contains 1 or 0 magnetic microsphere. Because of the low concentrations of IL-2 and IL-6 cytokines detected, most of the sphere surface did not form complete complexes with the galactosidase label and did not produce the fluorescent signal amplified by the galactosidase label. A small portion of the sphere surface forms one or a few complete complexes with a galactosidase label, thereby amplifying the fluorescent signal. The proportion of the spheres generating fluorescent signals to the total microspheres is positively correlated with the concentration of the analyte.
Example 2
High-sensitivity simultaneous detection of two cytokines of IL10 and IFNr
1. Two kinds of fluorescent coding microsphere synthesis: styrene monomer, polymethyl methacrylate, initiator, crosslinking agent and acrylic acid oligomer are mixed in chloroform to prepare high polymer solution. Two 4.5mL portions of polymer solution were taken and added with 0.5mL of rhodamine and 0.5mL of fluorescein and 90mg of nano-magnetic particles for reaction solution 1 and reaction solution 2, and 0.5mL of rhodamine and 0.5mL of fluorescein and 90mg of nano-magnetic particles for reaction solution 2. And then respectively placing 1 and 2 into two reactors with 300mL of deionized water and surfactant, taking 1 as an example, uniformly dispersing the 1 solution into 10-micrometer liquid drops under the conditions of stirring, ultrasonic treatment and surface force adjustment, adding an initiator into the solution, heating to perform polymerization crosslinking reaction, slowly dissolving chloroform in each liquid drop into water and volatilizing, polymerizing and crosslinking monomers after 24 hours, and finally forming the polymer microsphere 1,2 with the same process. The surfaces of the two polymer microspheres are provided with carboxyl functional groups.
2. Microspheres 1 and 2 were coupled to IL-10 and IFNr capture antibodies, respectively, and 1 as an example, 1mg of microspheres were dispersed in 1ml PBS buffer, 5mg of EDC and 5mg of Sulfo-NHS were added, mixed well and maintained for 10 minutes, washed with 1ml PBS, and 50ug of IL-2 capture antibody was added. Incubate for 30 minutes to 3 hours at room temperature with shaking. After adding a blocking agent such as BSA, the mixture was incubated at room temperature for 30 minutes with shaking, and the microspheres 1 were washed by magnetic separation and finally dispersed in PBS. Mixing the loaded magnetic beads 1 and 2 in equal volume, wherein the concentration of each magnetic bead is 0.5mg/ml
3. IL10 and INFr standards were spiked to 10% bovine serum at concentrations of (0,0.001,0.005,0.01,0.3,1.0, and 10 pg/ml), respectively. 100ul of each standard was taken, 50ul of the medium magnetic bead mixed solution was added, and after incubation for 3 hours with 25C shaking, washed three times with 5 XPBS+0.1% Tween. Dispersing into 100ul PBS, adding 50ul 1ug/ml IL-10 detection antibody and 50ul 1ug/ml IFNr detection antibody, incubating for half an hour at 25C, and washing with 5XPBS+0.1% Tween three times. Dispersing into 100ul PBS, and adding 50ul streptavidin-beta-galactosidase conjugated enzyme labeling reagent. Wash four times with 5x pbs+0.1% tween. Dispersed into 100ul of enzyme substrate solution. Samples containing unknown concentrations of the analyte are treated in the same manner.
4. Adding the microsphere composite structure into a reactor with a micropore plate chip through microfluid, adding 10MHz alternating current, pushing the microsphere into the micropore, sealing the surface of the micropore by silicone oil, reacting for 2 minutes, using 488nm wavelength light to excite, photographing an optical filter 1, photographing an optical filter 2, using 532 nm wavelength light to excite, photographing an optical filter 3, flowing in ethanol, flowing in cleaning liquid, changing the alternating current frequency to 10kHz, flowing in cleaning liquid, and cleaning the reactor.
5. Data processing and concentration determination. The image processing software first identified all the beads and classified the beads according to the intensity of fluorescence under filter 1 and filter 2 (bead 1, with IL-2 capture antibody and bead 2, with IL-6 capture antibody). The ratio of the total number of the labeling enzyme molecules to the total number of the magnetic beads was calculated for each concentration of each magnetic bead (AEB, average Enzyme per Bead). When the concentration of the analyte is low, the ratio of the magnetic beads generating the chemical signal to the total magnetic beads is detected (fon < 0.5), aeb= -ln (1-fon)). When fon >0.5, aeb=ibead/ibenzyme, ibead is the average of all magnetic bead generated signals in a single test, ibenzyme is the average of single enzyme molecule generated signals, ibenzyme=fon×ibead/AEB, aeb= -ln (1-fon), data for all fon <0.5 samples were used for calculation. And after the AEB value and the concentration value of the object to be measured are subjected to linear fitting to draw a calibration curve, testing the unknown sample by using the same method, and taking the obtained AEB value into a standard curve by using an interpolation method to obtain the concentration of the object to be measured in the unknown sample. IL-10 concentration was measured to be 10pg/ml and INFr concentration was measured to be 0.3pg/ml.
6. Determination of detection Limit
And (3) measuring the sample with the concentration of 0 for 10 times in parallel, and taking the standard deviation of the average value of the measured AEB value added with 3 times into a standard curve of 4. The solubility of the to-be-measured object obtained in the standard curve is the detection limit of the method. The limit of detection of IL-10 was measured to be 0.027pg/ml and the INFr limit of detection was measured to be 0.024pg/ml.
FIG. 2 shows the signal of the marker enzyme (galactosidase) detected by the forward scatter imaging module after oil sealing when the fluorescent encoding microspheres modified with sandwich complexes were introduced into a microplate when samples of IL-10 and IFNr cytokines of unknown concentration were measured in example 2. Each microwell contains 1 or 0 magnetic microsphere. Because of the low concentrations of IL-2 and IL-6 cytokines detected, most of the sphere surface did not form complete complexes with the galactosidase label and did not produce the fluorescent signal amplified by the galactosidase label. A small portion of the sphere surface forms one or a few complete complexes with a galactosidase label, thereby amplifying the fluorescent signal. The proportion of the spheres generating fluorescent signals to the total microspheres is positively correlated with the concentration of the analyte.
In conclusion, the multiplex immunodetection method and the kit provided by the invention can enable immune molecule detection to reach the femtocells (fg/ml), embody ultra-high sensitivity, and are matched with amplification-free nucleic acid molecule diagnosis equipment, so that the detection steps are simple, and the detection results are accurate and reliable.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.