Patent Application
20250093344
The present invention patent belongs to the field of biological analysis. Specifically, it relates to a digital immunoassay detection and analysis method for precise quantification of proteins.
For the determination of new biomarkers, enzyme-linked immunosorbent assay (ELISA) is no longer sufficient. For example, the sensitivity of this method is no longer sufficient for quantitative analysis of proteins such as P-tau-181, 217, 231 antigen and neurofibrillary light chain (NfL) associated with Alzheimer's disease. Although these have been included in clinical guidelines, traditional detection methods cannot achieve precise measurements due to the low levels of these substances in the blood. In addition, due to the large reaction system, the response of the reporter molecule is limited. The concentration of reporter molecule is too low, while the reaction system is too large, resulting in the effective signal concentration being diluted by the reaction solution.
In recent years, digital immunoassay method has received increasing attention due to its high sensitivity and is regarded as the new gold standard. For example, Quanterix's Simoa platform is a typical representative of the method. Firstly, the Simoa platform utilizes over 200,000 micron sized micropores as detection and sampling carriers, limiting the reaction system to the fL level, which is 1,000,000,000 times smaller in volume than the uL level of traditional ELISA methods. In this way, the signal generated by a reporter molecule is limited to a smaller system, and its concentration can reach the range that the instrument optical system can easily respond to (mg/mL level), and the number of observed molecules can be obtained by counting.
However, due to the inability of digital immune signals to sample all molecules and technical constraints or sample concentration issues, it cannot be guaranteed that all molecules can be sampled and detected. In other words, it cannot be guaranteed that every molecule can be ‘captured’ or ‘read’. In this case, the sampling results will be affected by randomness, which means that only some molecules can be sampled and detected, and there is a certain degree of uncertainty as to whether these molecules can represent the whole. At low concentrations, the sampling process follows Poisson Distribution, and a maximum of 200,000 micropores can only satisfy about 1-6000 digital immune signal conditions, with a number of digital immune signal wells greater than 99%. In addition, due to the presence of Poisson Distribution noise, the theoretical CV is ≥10% (or 20% if n is 25 or less), when the number of positive molecules (n) detected in the digital immunizations is less than 100. This determines that its linear range is narrow and can only be achieved in the state of 25-6000 molecules, which is difficult to meet the full digital requirements of practical applications.
In addition, the Simoa platform uses magnetic beads as capture units, and then introduces the magnetic beads into micropores for analysis. This process essentially involves secondary sampling, which undoubtedly increases the uncertainty of sampling. Due to the low efficiency of secondary sampling, according to relevant literature reports, the sampling efficiency of the Simoa platform HD-1 is only about 5%. This is mainly due to the limitation of the number of micropores by the chip area, as well as the efficiency issue caused by the introduction of magnetic beads into micropores.
Therefore, to improve the performance of digital immune detection, the key lies in increasing the number of micropores and improving the efficiency of secondary sampling. Although digital immune detection has enormous potential value, the high cost of the Simoa scheme makes it difficult to improve detection throughput and the narrow linear range makes it difficult to meet the needs of large-scale and low-cost applications.
At present, the method for digital detection by using micro droplets has been widely applied in digital PCR technology. The digital PCR systems on the market mainly use micro droplet generation technology, among which Bio-Rad's QX200 Droplet Digital PCR System is a typical representative. This system generates micro droplets through microfluidic chips or microfluidic valves, and each micro droplet can be seen as a micro reaction chamber that can perform independent PCR reactions. In Bio-Rad's system, the generation of micro droplets is achieved through a technique called “water-in-oil”, which involves adding surfactants to two immiscible liquids (such as water and oil) and forming micro droplets through physical shear or pressure driving. The size of each micro droplet is about 1 nanoliter, and each PCR reaction can generate approximately 20,000 micro droplets. These micro droplets are evenly distributed in the reaction tube during the PCR reaction, so that the number of DNA molecules in each micro droplet follows Poisson Distribution, thereby achieving digital PCR detection.
This technology has been applied in some fields. However, in contrast, digital immune detection based on micro droplets has not yet been commercially applied.
Therefore, it is of great significance to develop a highly sensitive, accurate, and efficient digital immunoassay detection method in this field.
Provided is a highly sensitive, accurate, and efficient digital immunoassay detection method.
The first aspect of the present invention provides a digital immunoassay detection and analysis method for precise quantification of proteins, comprising the steps of:
In another preferred embodiment, the method further comprises the following sub steps in step (s1):
In another preferred embodiment, the particle size of the magnetic bead is 0.03-100 μm.
In another preferred embodiment, the particle size of the magnetic bead is 1-5 μm.
In another preferred embodiment, the method further comprises the following sub steps in step (s3):
In another preferred embodiment, the method further comprises the following steps between steps (s3) and (s4): washing the sheared water-in-oil second mixture to remove empty droplets that do not contain any first or second micro droplets.
In another preferred embodiment, the method further comprises the step in step (s4): measuring the second micro droplet to obtain the number of the second micro droplet and the ratio of the number of the first micro droplet to the second micro droplet, thereby obtaining a quantitative detection result of the proteins.
In another preferred embodiment, the method further comprises the following step in step (s2):
In another preferred embodiment, the oil phase is a fluorinated oil selected from the group consisting of: HFE7500, FC40, Fluo-Oil 7500, Fluo-Oil 40, Fluo-oil 135, Fluo-Oil 200, and a combination thereof.
In another preferred embodiment, a fluorinated surfactant needs to be added to the fluorinated oil to form the final oil phase.
In another preferred embodiment, the fluorinated surfactant is selected from the group consisting of: fluoroalkyl polyether sulfate, fluoroalkyl polyethylene glycol ether sulfate, fluorocarbon-based alcohol and fluorocarbon-based acid, PFPE-PEG polymer, PFPE-b-PPO-PEO-PPO-b-PFPE, and a combination thereof.
In another preferred embodiment, the enzyme labeled molecule is streptavidin-HRP.
In another preferred embodiment, the washing is carried out by adsorbing and intercepting micro droplets wrapped with individual magnetic beads through magnetic force, thereby washing and enriching the magnetic beads.
In another preferred embodiment, the magnetic bead is a magnetic bead with fluorescent properties or a magnetic bead without fluorescent properties.
In another preferred embodiment, the magnetic bead is a magnetic bead with fluorescent properties.
In another preferred embodiment, the method uses multi-color magnetic beads with fluorescent properties for quantitative detection of multiple substances.
In another preferred embodiment, wherein the method is used for measuring n proteins, and the value of n is 1-200, preferably 1-20, and more preferably 1-10.
In another preferred embodiment, when detecting n proteins, the second mixture contains the first micro droplets corresponding to each protein.
In another preferred embodiment, the total number of the first and second micro droplets in the second mixture effectively quantified by the method is greater than 50,000.
In another preferred embodiment, the total number of the first and second micro droplets in the second mixture effectively quantified by the method is ranging from 50,000 to 1 million.
In another preferred embodiment, the total number of the first and second micro droplets in the second mixture effectively quantified by the method increases with the expansion of the optical camera target surface.
In another preferred embodiment, the magnetic beads with fluorescent properties emit fluorescence at different wavelengths, comprising green (520 nm), yellow (490 nm), red (610 nm), blue (450 nm), as well as rare earth europium fluorescence and rare earth terbium fluorescence.
In another preferred embodiment, the magnetic beads further have different modifications.
In another preferred embodiment, the magnetic bead modification comprises carboxyl, amino, p-toluenesulfonic acid, NHS, Biotin, and streptavidin (SA).
In another preferred embodiment, the magnetic bead is a magnetic bead without fluorescent properties.
In another preferred embodiment, when the magnetic bead is a magnetic bead without fluorescent properties, step (s1f) is used to fluorescently label the magnetic beads.
In another preferred embodiment, the antibody is conjugated with carboxyl groups in step (s1b).
In another preferred embodiment, in step (s3), a second mixture containing more than 10,000 micro droplets is rapidly formed during mechanical shear treatment of the first mixture.
In another preferred embodiment, in step (s3), a second mixture containing 5-10 billion micro droplets is rapidly formed during mechanical shear treatment of the first mixture.
In another preferred embodiment, in step (s3), the volume of the water-in-oil second mixture is 10-1,000 uL.
In another preferred embodiment, the total volume of micro droplets in the second mixture is 10-1,000 uL.
In another preferred embodiment, the concentration of micro droplets in the second mixture is 0.5-90% (v/v).
In another preferred embodiment, the size of the micro droplet is 1-100 μm.
In another preferred embodiment, the mechanically shearing is carried out by homogenizing the first mixture using a high-speed homogenizer.
In another preferred embodiment, the speed of the high-speed homogenizer is ranging from 2,000 to 100,000 RPM.
In another preferred embodiment, the speed of the high-speed homogenizer is 10,000 RPM.
In another preferred embodiment, the ratio D1/D2 of the diameter D1 of the first micro droplet to the diameter D2 of the magnetic bead contained therein is 1.2-20, preferably 1.5-5, more preferably 1.6-4.
In another preferred embodiment, the ratio D1/D2 of the diameter D1 of the first micro droplet to the diameter D2 of the magnetic bead contained therein is 1.8-3.
In another preferred embodiment, the total content of the first and second micro droplets in the water-in-oil second mixture is less than 30%, preferably less than 20%, and more preferably less than 10%, relative to a total content of empty micro droplets in the second mixture.
In another preferred embodiment, the content of the first micro droplets in the water-in-oil second mixture is greater than 30%.
In another preferred embodiment, the chromogenic substrate comprises ADHP (OxiRed probe), BBTP, and FDG.
In another preferred embodiment, the chromogenic substrate is ADHP (OxiRed probe).
In another preferred embodiment, there is no cross interference between the spectrum of the substance produced by the enzymatic reaction between the positive complex and the chromogenic substrate and the emission wavelength of the negative complex.
In another preferred embodiment, in step (s3b), the conditions for the static reaction are: reacting at 37° C. for 5 minutes.
In another preferred embodiment, in step (s4), the fluorescence signals of the green and red channels are used to accurately count the number of the first and second micro droplets after the reaction.
In another preferred embodiment, in step (s4), a droplet imaging instrument is used for fluorescence imaging, which can capture the number of first micro droplets n and the number of second micro droplets m within one detection cycle.
In another preferred embodiment, in step (s4), the droplet imaging instrument uses a monochrome or color camera, which is a CCD camera or CMOS camera with more than 5 million pixels, or a monochrome or color camera.
In another preferred embodiment, in step (s4), a special low imaging and low distortion lens is selected to enable clear resolution of a magnetic bead.
In another preferred embodiment, in step (s4), the measurement comprises measuring the second micro droplet by using a fluorescent antibody to bind to a magnetic bead—antibody complex and labeling the magnetic bead with a fluorescent label (negative signal label); alternatively, magnetic beads with fluorescent properties can be selected, based on the characteristic of no cross interference between emission wavelength thereof and the spectrum of the substance produced by the enzymatic reaction in step (s3), in order to achieve the determination of the second micro droplet.
In another preferred embodiment, when the magnetic bead is a magnetic bead with fluorescent properties, the fluorescence of the magnetic bead can produce different fluorescence intensities depending on the content of fluorescent dyes, thereby further identifying and resolving the magnetic bead.
The second aspect of the present invention provides a digital immunoassay device for quantification of proteins, wherein the device comprises:
In another preferred embodiment, the particle size of the magnetic bead is 0.03-100 μm.
In another preferred embodiment, the particle size of the magnetic bead is 1-5 μm.
In another preferred embodiment, the magnetic bead is a magnetic bead with fluorescent properties or magnetic beads without fluorescent properties.
In another preferred embodiment, the oil phase is a fluorinated oils selected from the group consisting of: HFE7500, FC40, Fluo-Oil 7500, Fluo-Oil 40, Fluo-oil 135, Fluo-Oil 200, and a combination thereof.
In another preferred embodiment, a fluorinated surfactant needs to be added to the fluorinated oil to form the final oil phase.
In another preferred embodiment, the fluorinated surfactant is selected from the group consisting of: fluoroalkyl polyether sulfate, fluoroalkyl polyethylene glycol ether sulfate, fluorocarbon-based alcohol and fluorocarbon-based acid, PFPE-PEG polymer, PFPE-b-PPO-PEO-PPO-b-PFPE, and a combination thereof.
In another preferred embodiment, in step (s3), the volume of the water-in-oil second mixture is 10-1,000 uL.
In another preferred embodiment, the concentration of micro droplets in the second mixture is 0.5-90% (v/v).
In another preferred embodiment, the size of the micro droplets is 1-100 μm.
In another preferred embodiment, the mechanically shearing is carried out by homogenizing the first mixture using a high-speed homogenizer.
In another preferred embodiment, the speed of the high-speed homogenizer is ranging from 2,000 to 100,000 RPM.
In another preferred embodiment, the speed of the high-speed homogenizer is 10,000 RPM.
In another preferred embodiment, the ratio D1/D2 of the diameter D1 of the first micro droplet to the diameter D2 of the magnetic bead contained therein is 1.2-20, preferably 1.5-5, more preferably 1.6-4.
In another preferred embodiment, the ratio D1/D2 of the diameter D1 of the first micro droplet to the diameter D2 of the magnetic bead contained therein is 1.8-3.
In another preferred embodiment, a total content of the first and second micro droplets in the water-in-oil second mixture is less than 30%, preferably less than 20%, and more preferably less than 10%, relative to a total content of empty micro droplets in the second mixture.
In another preferred embodiment, the chromogenic substrate is ADHP (OxiRed probe).
In another preferred embodiment, in step (s4), a droplet imaging instrument is used for fluorescence imaging, which can capture the number of first micro droplets n and the number of second micro droplets m within one detection cycle.
In another preferred embodiment, the method is used to determine n proteins, and the value of n is 1-200, preferably 1-20, and more preferably 1-10.
In another preferred embodiment, when detecting n proteins, the second mixture contains the first micro droplets corresponding to each protein.
In another preferred embodiment, when detecting n proteins, the second mixture contains the first micro droplets corresponding to each protein.
In another preferred embodiment, the total number of the first and second micro droplets in the second mixture effectively quantified by the method is greater than 50,000.
In another preferred embodiment, the total number of the first and second micro droplets in the second mixture effectively quantified by the method is ranging from 50,000 to 1 million.
It should be understood that within the scope of the present invention, each technical feature of the present invention described above and in the following (such as examples) may be combined with each other to form a new or preferred technical solution, which is not listed here due to space limitations.
Through extensive and deep research, the inventor has developed for the first time a highly sensitive, accurate, and efficient digital immunoassay method for precise quantification of proteins. Specifically, it comprises the following steps: (s1) providing an aqueous phase mixture comprising the following complexes: (A) a positive signal complex: a magnetic bead—antibody—antigen—detection antibody—enzyme label, and/or a magnetic bead—antibody—antigen—detection antibody complex; and (B) a negative signal complex: a magnetic bead—antibody complex; (s2) mixing the aqueous phase mixture with a chromogenic substrate and an oil phase to form a first mixture; (s3) mechanically shearing of the first mixture to form a sheared water-in-oil second mixture; wherein, the second mixture comprises a first micro droplet and a second micro droplet; the first micro droplet is a micro droplet containing a positive signal complex; the second micro droplet is a micro droplet containing a negative signal complex; and (s4) measuring the first micro droplet to obtain the number of the first micro droplets and obtain the quantitative detection result of the proteins. On this basis, the present invention has been completed.
As used herein, the abbreviation “HRP” refers to horseradish peroxidase, which can bind with streptavidin to form a stable complex.
As used herein, the abbreviation “ADHP” is a highly sensitive and stable peroxidase substrate that selectively detects hydrogen peroxide (H2O2). In the presence of horseradish peroxidase (HRP) system, this substantially colorless and non-fluorescent ADHP reacts with H2O2 in a 1:1 chemical quantitative ratio to generate strong red fluorescence of Resorufin, which can be analyzed by fluorescence spectrophotometer and Microplate reader. Many enzyme reactions can produce H2O2, therefore ADHP can be used to detect the biological activity of many different enzymes in coupled enzymatic reactions.
As used herein, “microsphere” and “magnetic bead” can be used interchangeably, referring to a magnetic bead with or without fluorescent properties in the present invention.
As used herein, “the first antigen of the present invention”, “the antigen of the present invention”, and “the protein to be tested of the present invention” can be used interchangeably, referring to the protein to be accurately quantified.
As used herein, the term ‘comprising’ can be open, semi closed, or closed. In other words, the term includes the following limitations: “containing”, “substantially composed of”, “composed of”.
Enzyme linked immunosorbent assay (ELISA) is a commonly used protein quantitative analysis method. Since its introduction in the 1970s, this technology has been widely used in various scientific research and has become an indispensable classic technique in the field of biomedical research and development. ELISA is a basic immunoassay method that utilizes enzymatic reactions to quantitatively analyze the binding of antigens to antibodies, and it is used for the identification of peptides, proteins, antibodies, hormones, and so on. Over the past few decades, due to its high sensitivity and high-throughput, it has been the preferred technique for protein quantitative analysis and is widely recognized as the “gold standard” in the industry. The basic reaction process involves the specific binding of antibody-coated well plates with antigen in the sample, followed by the specific binding of detection antibodies labeled with reporter molecules to the antigens. Finally, the signals generated by the reporter molecules, such as color reactions, optical reactions, magnetic reactions, or electrochemical reactions, are used to indirectly quantify and analyze the antigens. Although this method has certain advantages, there are also some unavoidable drawbacks. The biggest issue is that it is essentially still a simulated quantitative testing technology, which can no longer meet the needs of measuring new biomarkers.
Unlike molecular hybridization reactions that only involve the interaction of two molecules (probe and target), immune reactions typically involve multi-faceted interactions of antigens, antibodies, and auxiliary molecules.
Therefore, in order to effectively carry out immune reactions, it is necessary to use carriers containing antibodies to capture and enrich target proteins. This method has been widely used in various immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).
Secondly, regarding the requirement for signal amplification. In immune detection, due to the extremely small number of target molecules, additional reporter molecules need to be introduced to amplify the detection signal. This is usually achieved by introducing fluorescent or enzyme labeled reporter molecules, which can bind to target molecules or antibodies to generate detectable signals.
Finally, it is necessary to consider the characteristics of Poisson Distribution. In droplet microfluidic systems, each droplet can be considered as an independent reaction chamber, therefore, the number of molecules in each droplet should follow Poisson Distribution.
In digital immunoassay detection of droplets, the Poisson Distribution formula can be used to describe the distribution of target molecules in droplets. The Poisson Distribution formula is as follows:
However, it is necessary to control the number of target molecules containing two or more molecules in the droplet, that is, the number when k>1, such as λ=0.1, k=1, P1≈0.090, the cumulative probability of k>1 P1-∞≈0.0046, P1/P1-∞≈20, that is, when λ=0.1, the ratio of the number of droplets containing only one molecule to the number of droplets containing two or more molecules is 20, and the probability of droplets with K=0 is 0.90, which can be understood as 90% of droplets being empty droplets, 9% of droplets containing only one molecule, and 0.47% of droplets containing two or more molecules. The present invention sets the above conditions as the minimum λ standard. The presence of empty droplets greatly reduces the number of effective droplets containing magnetic beads that can be observed in the end. To improve the efficiency of observing magnetic beads, effective measures should be taken to eliminate empty droplets.
λ is the average number of target molecules, which is the ratio of the number of detected molecules (n) to the detection unit (m). Therefore, when λ≤0.1, effectively increasing the number of m is the key technology for achieving digital detection.
In addition to the characteristics of Poisson Distribution, digital immunoassay detection of droplets also needs to consider other factors, such as antibody capture and enrichment, signal amplification, etc.
By comprehensively considering these factors, the inventor has designed an efficient digital immunoassay detection method for droplets, thereby achieving sensitive detection of target molecules.
These are the key factors that need to be considered in droplet based digital immunoassay detection, and only by meeting these conditions can effective digital immunoassay detection be achieved.
The present invention utilizes micro droplet technology to disperse magnetic beads into water-in-oil micro droplets within 1-10 seconds through mechanical emulsification dispersion method. Magnetic beads will be randomly encapsulated in micro droplets to form water-in-oil droplets. The number of magnetic beads in the micro droplets follows Poisson Distribution, and then magnetic force is used to wash and trap the micro droplets containing magnetic beads, separating the empty droplets and significantly increasing the number of micro droplets containing magnetic beads that can be observed.
The present invention chooses to use monodisperse magnetic beads with a particle size of 1-5 μm. These magnetic beads can be widely used in the field of life sciences, including products from companies such as Dynabeads, MagSi, MyOne, M-270, Dynal Biotech, Sera-Mag, etc. The particle size range of these magnetic beads is from 0.03 μm to 100 μm. The magnetic beads used in the present invention have or do not have fluorescent properties, and can emit light of different wavelengths such as green (520 nm), yellow (490 nm), red (610 nm), blue (450 nm), as well as rare earth europium fluorescence and rare earth terbium fluorescence. These magnetic beads also have different modifications, including carboxyl, amino, p-toluenesulfonic acid, NHS, Biotin, and streptavidin (SA).
The present invention uses fluorinated oil as the oil phase, which is widely used in the field of life sciences. Common fluorinated oils include HFE7500, FC40, Fluo Oil 7500, Fluo Oil 40, Fluo Oil 135, and Fluo Oil 200. Adding fluorinated surfactants to fluorinated oil can form an oil phase. Common surfactants include fluoroalkyl polyether sulfate, fluoroalkyl polyethylene glycol ether sulfate, fluorocarbon-based alcohol and fluorocarbon-based acid, PFPE-PEG polymer, PFPE-b-PPO-PEO-PPO-b-PFPEs, etc. After mixing the above oil phase with aqueous phase, water-in-oil micro droplets can be rapidly formed using a high-speed homogenizer.
The aqueous phase of the present invention is a magnetic bead solution. By adding the magnetic bead solution to the oil phase solution and using a high-speed homogenizer, the magnetic beads can be dispersed into monodisperse water-in-oil droplets, with a controlled number of 500,000 magnetic beads. Using 100 μL of aqueous phase, it is estimated that millions to billions of micro droplets can be generated each time. Under these conditions, the magnetic beads are present in the form of a single bead encapsulated within a single microdroplet. Due to the excellent properties of surfactants in water-in-fluorinated oil, micro droplets can be present stably in the oil phase without leaking fluorescent substances to each other.
The water-in-oil magnetic beads can temporarily and stably adsorb onto the glass slide after being attracted by a magnet. The remaining droplets that do not contain magnetic beads are washed away by washing with the oil phase. At this time, the water-in-oil magnetic beads form a monodisperse magnetic bead layer on the glass slide, which can be observed by fluorescence imaging. A single magnetic bead can be clearly distinguished.
The present invention selects fluorescence microscope lenses with low deformation and low dispersion, with priority given to 1×, 2×, 4×, 10×, 20×, 40×, 60×, and 100× objectives. By adjusting the optical configuration, ensure that the formed phase field can observe the fluorescence signals generated by the magnetic beads and substrate. The present invention uses a dual channel fluorescent filter, which includes at least two channels selected from the group consist of: a DAPI channel with an emission wavelength of about 450-500 nm; a FITC channel with an emission wavelength of approximately 515-530 nm; a GFP channel with an emission wavelength of approximately 509-525 nm; Alexa Fluor 488 channel with an emission wavelength of approximately 519-545 nm; Cy3 channel with an emission wavelength of approximately 565-615 nm; Rhodamine Red-X channel with an emission wavelength of approximately 600-680 nm; Texas Red channel with an emission wavelength of approximately 600-680 nm; Cy5 channel with an emission wavelength of approximately 665-725 nm. The present invention uses a CCD or CMOS camera to capture fluorescent images, with the target size of the selected camera matching the lens. For example, cameras in sizes such as 4/3 inches, 1.2 inches, 1.1 inches, 1 inch, 1/1.1 inches, 1/1.2 inches, ⅔ inches, 1/1.8 inches, ½ inches, ⅓ inches, 36/24 millimeters, 45/60 millimeters, and 60/60 millimeters can be used. The pixel count of a camera can range from 1 million to 100 million pixels. The pixel size should match the numerical aperture (NA) of the selected objective lens. For example, when using a 1× objective lens with an NA of 0.045, according to the Rayleigh criterion (0.61λ/NA), its resolution at a wavelength of 500 nm is approximately 4.35 um. Therefore, the present invention can choose a pixel size of 3.45 um (⅔ inch camera) that matches it. In the case of 2-5 um magnetic beads, a pixel bright spot on the phase field can represent a magnetic bead signal.
After the immune reaction, mix the magnetic beads with the enzyme substrate at low temperature (2-8° C.) evenly, then immediately add the oil phase reagent and use a high-speed homogenizer for 1s-60s dispersion. This can evenly disperse the magnetic beads into the micro droplets. Due to the fact that the number of empty droplets is much larger than the number of magnetic beads, according to the Poisson Distribution principle, magnetic beads will be individually and uniformly dispersed into a single droplet. The size of magnetic bead droplets will be larger than the diameter of the magnetic beads, and the measured droplet size is between 1-10 times the size of the magnetic beads. The size of droplets is mainly affected by the speed of the high-speed homogenizer, the content of oil phase surfactants, and the action time of the high-speed homogenize. The typical droplet size is between 1-2 times the size of magnetic beads. Due to the shear force, the droplet size follows a normal distribution. In order to control the uniform distribution of droplets as much as possible, the reaction system parameters can be adjusted, including the speed of high-speed homogenizer, the content of oil phase surfactants, and the length of the high-speed homogenizer action time. During the color development process after the reaction, enzyme fluorescent substrates are used for color development. Commonly used substrates include ADHP (OxiRed probe) for horseradish peroxidase, BBTP for alkaline phosphatase, and FDG substrate for galactosidase.
The final resulting first microdroplets are characterized by containing both fluorescent magnetic beads and fluorescent substrate coloration, and they satisfy the following conditions simultaneously: 1) droplets containing only fluorescent magnetic beads; 2) droplets containing fluorescent magnetic beads and also exhibiting fluorescence from enzymatic substrate coloration; 3) excluding microdroplets that solely exhibit fluorescence from substrate coloration without containing fluorescent magnetic beads. All three conditions must coexist.
Magnetic beads are coupled with a certain proportion of capture antibodies, and after washing the beads, they are blocked with bovine serum albumin (BSA) solution. Followed by another wash, the resulting magnetic bead—antibody complexes are prepared for subsequent use. The concentration of these magnetic bead—antibody complexes is determined by counting, confirming that there are 500,000 beads. With the number of magnetic beads controlled, the reaction system is controlled to 50 μL. The above magnetic beads are allowed to react with the antigen for 15 minutes, then washed three times with a magnetic frame PBST 200 μL. Afterward, biotinylated detection antibody solution is added, and the mixture is allowed to react for 15 minutes, then washed three times with a magnetic frame PBST 200 μL. Afterward, streptavidin-HRP is added, and the mixture is allowed to react for 15 minutes, then washed three times with a magnetic frame PBST 200 μL. Afterward, 10 μL of anti-mouse IgG-FITC is added, and the mixture is agitated for 10 minutes, then washed three more times with a magnetic frame PBST 200 μL. The above magnetic beads are configured into a 100 uL solution (50 uL pre-cooled PBS with 50 uL pre-cooled ADHP) using pre-cooled PBS (2-8° C.) and the reaction is shown in
After the above magnetic bead reaction solution is fully prepared, the liquid mixture is injected quickly into 100 L of oil for droplet generation at once, and dispersed using a high-speed homogenizer for 1-60s to form a water-in-oil droplet mixture. And the droplet mixture is reacted at 37° C. for 5 minutes, and the above droplets are washed with a magnetic frame to remove unreacted empty droplets.
The above micro droplet solution is added to the detection cell for counting, and all green channel highlight counts n, and all red channel highlight counts m are recorded. The static detection cell consists of a fluorescence counting plate with a 100 μm sandwich in the center. The green and red channels, with wavelengths of 570 excitation, 585 nm emission and 470 excitation and 490 emission, are detected without interference. The detection method is shown in
Multi colored fluorescent microspheres can also be used for multi marker detection. To achieve this, the instrument settings are adjusted, one color and multiple fluorescent microspheres with different intensities are employed as carriers, and each color and intensity of magnetic beads are coupled with a certain proportion of different capture antibodies. After cleaning the magnetic beads, they are washed and blocked with bovine serum albumin solution. After washing, magnetic bead—antibody complexes with different fluorescence intensities will be formed for subsequent use. The formed antibody coated magnetic beads are counted, and the concentration of magnetic beads is determined to be greater than 50,000, and then the magnetic beads are mixed. The total volume of magnetic beads is controlled and the reaction system is controlled to 50 uL, the above magnetic beads are reacted with antigens for 15 minutes, then washed three times with 200 uL of PBST using a magnetic frame. Afterward, biotinylated detection antibody solution is added, and the mixture is allowed to react for 15 minutes, then washed three times with 200 μL of PBST using a magnetic frame. Afterwards, streptavidin-HRP is added, and the mixture is allowed to react for 15 minutes, then washed three times with 200 L of PBST using a magnetic frame. The above magnetic beads are configured into a 100 uL solution (50 uL pre-cooled PBS with 50 uL pre-cooled ADHP) using pre-cooled PBS (2-8° C.) and the reaction is shown in
A fluorescent microsphere can also be used for rapid biomarker detection. To achieve this, the instrument settings are adjusted, a colored fluorescent microsphere is employed as a carrier, magnetic beads are coupled with a certain proportion of different capture antibodies. After cleaning the magnetic beads, they are washed, blocked with bovine serum albumin solution, and magnetic bead—antibody complexes are formed for subsequent use. The antibody-coated magnetic beads are counted to determine the concentration of magnetic beads to be greater than 50,000 beads and then the magnetic beads are mixed. The total volume of magnetic beads was controlled to 50 uL, the above magnetic beads are reacted with the antigen for 5 min, then washed three times with 200 uL of PBST using a magnetic frame. Followed by the addition of HRP detection antibody solution, the magnetic beads are allowed to react for 5 min, then washed three times with 200 uL of PBST using a magnetic frame. And the above beads were configured into a 100 uL solution using pre-cooled PBS (2-8° C.) (50 uL pre-cooled PBS with 50 uL pre-cooled ADHP) and the reaction is shown in
The process of the micro droplet magnetic bead algorithm of the present invention is as follows:
The main advantages of the present invention include:
The present invention will be further illustrated below with reference to the specific examples. It should be understood that these examples are only for illustrating the present invention and not intend to limit the scope of the present invention. Experimental methods not specified in the following examples are usually under conventional conditions, such as conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, percentages and portions are weight percentages and weight portions.
The experimental materials used in this example are shown in Table 1.
The stratification of the mixed solution and the resulting homogenate are shown in
Here, if the reaction is carried out first before mechanically hearing, it will cause an increase in the fluorescence of the empty droplets, which is not effective.
Regression analysis between the concentration of the calibration and the percentage of positive magnetic beads in Table 3 showed a linear relationship within the range of 1-1000 fg/mL of calibration concentration, which had a good correlation with the calculated values based on Poisson's theory.
Tables 4-5 shown that the CV of the 5fg/mL sample measured was 7.54%, and the CV of 100fg/mL was 0.01%.
The results in Table 6 indicated that the method of the present invention can effectively detect IL6 in serum.
The experimental materials for this example are shown in Table 7
The magnetic bead coating method of this example was EDC coupling method, and the specific operation steps were as follows:
Activation solution 1: 100 mg/mL EDC was prepared: 10 mg EDC was weighed and added with 100 uL pre cool 100 mM MES at pH 5.0, dissolved.
Activation solution 2: 100 mg/mL NHS was prepared: 20 mg NHS was weighed and added with 200 uL pre cool 100 mM MES at pH 5.0, dissolved.
The amount was prepared according to demand.
For example, magnetic beads: antibody=50:1 for coupling, with a total volume of no less than 250 uL as shown in Table 9. The concentrations of capture antibodies for IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, and IL-15 were calculated according to the instructions provided by the respective manufacturers, and preservatives, glycerol, and other components were removed by dialysis. The mixture was mixed well and combined at room temperature for 60 minutes.
Table 10 shows that effective quantitative analysis of multiple cytokines (IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15) can be performed using seven fluorescent magnetic beads with different green fluorescence intensity, with a detection range of 0-500 fg/mL.
The experimental materials for this example are shown in Table 11.
Note: The final concentration of ammonium sulfate in the coating solution is 1M/L, and the minimum coating volume should not be less than 300 uL.
The fluorescence images under two excitation conditions: excitation at 570 nm and emission at 585 nm, and excitation at 470 nm and emission at 490 nm were analyzed. And the number of fluorescent droplets or magnetic beads under the two channels was counted. Droplets that did not contain magnetic beads were blank droplets with no fluorescence.
Table 13 shows that using the method of this example to detect SARS-CoV-2 antigen standard can quantitatively detect the antigen in the range of 0-10000 fg/mL.
All references mentioned in the present application are incorporated by reference herein, as though individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, various changes or modifications may be made by those skilled in the art, and these equivalents also fall within the scope as defined by the appended claims of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202311209690.7 | Sep 2023 | CN | national |
