CN114706023A - Magnetic detection method for biomolecular interaction - Google Patents
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Abstract
The invention provides a magnetic detection method for biomolecular interaction, which comprises the following steps: a. labeling or capturing a biomolecular interaction pair in a biological sample by a superparamagnetic particle, thereby binding the biomolecular interaction pair on a functionalized diamond surface comprising an ensemble NV color center sensor; b. magnetically imaging the superparamagnetic particles on the surface of the diamond containing the ensemble NV color center sensor by using a wide-field magnetic imaging microscope; and c, establishing a mapping relation between the obtained magnetic image and the distribution of the superparamagnetic particles through a deep learning model, performing inverse solution and reconstruction on the distribution of the superparamagnetic particles, then counting the number of the superparamagnetic particles on the surface of the diamond, and analyzing the interaction of the target biomolecules according to quantitative data of the superparamagnetic particles. The invention can realize high sensitivity, low background, high stability and high specificity detection on the interaction of biological molecules, and the detection sensitivity can reach single molecule.
Description
Technical Field
The invention belongs to the field of magnetic detection, and particularly relates to a magnetic detection method for biomolecular interaction.
Background
The biomolecular interaction represented by protein-protein interaction is the key to normal progress of life activities, and the revealing of the biomolecular interaction has great significance for understanding complex physiological processes in a living body, screening and developing drugs, diagnosing and treating diseases, and the like. The detection of biomolecular interactions can be simply divided into in vivo and in vitro detection. For in vitro detection, Co-immunoprecipitation (Co-IP) is currently used as a gold standard for studying protein interaction under physiological conditions, and in addition, Surface Plasmon Resonance (SPR), single-molecule fluorescence, and other methods are widely used for detecting biomolecular interactions.
Co-IP is used for capturing and separating the protein interaction pair of interest in a sample to be detected through agarose beads or magnetic beads, and then analyzing the interaction through Western-Blot or mass spectrometry, so that the natural protein interaction can be analyzed, but the method has the defects of high sample demand, low sensitivity and only qualitative and semi-quantitative analysis. The Surface Plasmon Resonance (SPR) technique, which uses the change in optical refractive index caused by binding of biomolecules on a metal substrate to study interaction, can study the kinetics of interaction between biomolecules without labeling, however, the need for protein purification and the high cost of experimental equipment limit the application of this method. The monomolecular fluorescence technology is to monitor the biomolecules marked by fluorescence on a solution or a substrate by using a fluorescence microscope, can realize the quantitative identification of the biomolecular level interaction, does not need a purified sample, but has poor light stability of a fluorescent label, thereby limiting the long-term study on the protein interaction. Can not meet the requirements of the research on the interaction of biomolecules on basic scientific research and clinical diagnosis on the aspects of small sample demand, high sensitivity, specificity, stability and high efficiency.
The nano magnetic particles have the characteristics of high stability, low background, good biocompatibility and the like, and can be magnetically separated by utilizing an external magnetic field so as to realize the functions of magnetic purification, magnetic enrichment and the like. By utilizing the characteristics, the specificity and the sensitivity of biomolecule interaction detection can be effectively improved. In the prior art, only few methods such as Hall probes and giant magneto-resistance (GMR) can give consideration to both detection cost and sensitivity, and due to the defects of the methods in the spatial resolution capability, the methods can only research the interaction between biomolecules at the level of ensemble (the integral effect of a plurality of molecules) at present and lack single-molecule information.
Therefore, there is a need for methods that enable highly sensitive, low background, highly stable and highly specific detection of biomolecular interactions.
Disclosure of Invention
The recently developed nitrogen-vacancy color center (NV color center) of diamond has both high sensitivity and high spatial resolution for magnetism measurement in room temperature and atmospheric environment, and is a very attractive quantum magnetic sensor. The good biocompatibility of diamond makes this system suitable for biomedical applications. Aiming at the defects of the existing biomolecule interaction detection technology, the invention provides a novel biomolecule interaction in-vitro detection method using single-particle magnetic imaging. The invention uses diamond NV color center as substrate and sensor, magnetic particles as label, and uses wide field magnetic imaging microscope device for biological sample detection to image, identify and count the magnetic particles on the diamond surface, so as to realize high sensitivity, low background, high stability and high specificity detection of biological molecule interaction, and the detection sensitivity can reach single molecule.
Specifically, the invention aims to use the NV color center in the diamond as a sensitive magnetic sensor, and realize the magnetic detection technology aiming at the interaction of the biomolecules through the surface functionalization treatment of the diamond, the capture and magnetic labeling of the biomolecule pairs to be detected, wide-field magnetic imaging and magnetic image analysis. The invention has the advantages of low magnetic background, stable signal, single molecule visualization, high sensitivity and high specificity, can make up for the defects of the existing biomolecule interaction detection method to a great extent, and provides a powerful technology for the research of biomolecule interaction and related high-sensitivity biomedical detection.
In some embodiments, the diamond surface may capture the interaction pair to be detected in the sample by modifying and immunologically functionalizing a bulk diamond containing NV colour centers. Superparamagnetic particles are adopted to replace fluorescent labels widely applied at present to mark interesting biomolecule interaction pairs, and a signal source with low background and high stability is provided. By utilizing the controllable characteristic of the magnetic particles under an external magnetic field, magnetic enrichment and magnetic purification can be carried out according to needs to improve the sensitivity and specificity of detection. And performing magnetic detection on the wide-field magnetic microscope in a continuous spectrum mode, and performing single-particle magnetic imaging on the magnetic particles on the surface of the diamond. And (3) performing inverse solution and statistics on the single particle signals in the original magnetic field image by using an image processing method based on deep learning, and further obtaining quantitative information of the molecules to be detected.
The invention provides the following technical scheme:
in one aspect, the present invention provides a method for magnetic detection of biomolecular interactions comprising the steps of
a. Labeling or capturing a biomolecular interaction pair in a biological sample by a superparamagnetic particle, thereby binding the biomolecular interaction pair on a functionalized diamond surface comprising an ensemble NV color center sensor;
b. magnetically imaging the superparamagnetic particles on the surface of the diamond containing the ensemble NV color center sensor by using a wide-field magnetic imaging microscope; and
c. and establishing a mapping relation between the obtained magnetic image and the distribution of the superparamagnetic particles through a deep learning model, performing inverse solution and reconstruction on the distribution of the superparamagnetic particles, then counting the number of the superparamagnetic particles on the surface of the diamond, and analyzing the interaction of the target biomolecules according to quantitative data of the superparamagnetic particles.
In some embodiments, functionalization of the diamond surface containing ensemble NV color center sensors is achieved by biotin/streptavidin engineering.
In some embodiments, functionalizing the diamond surface containing the ensemble NV color center sensor further comprises passivating the diamond surface by pegylation modification.
In some embodiments, the functionalization of the diamond surface containing an ensemble NV color center sensor comprises hydroxylation, amination, passivation and biotin and streptavidin modification of the diamond surface containing an ensemble NV color center sensor, optionally further comprising treating the diamond surface containing an ensemble NV color center sensor with a biotinylated antibody.
In some embodiments, the hydroxylation is performed by a piranha solution.
In some embodiments, the amination is performed by an aminosilane.
In some embodiments, the amination is by an aminosilane: acetic acid: methanol in a volume ratio of 1-5:5:100, preferably 1: 5:100, respectively.
In some embodiments, passivation is performed by methylating polyethylene glycol succinimidyl valerate or methoxypolyethylene glycol active ester (mPEG-NHS).
In some embodiments, Biotin modification is performed by biotinylating polyethylene glycol succinimidyl valerate or biotinylating polyethylene glycol active ester (Biotin-PEG-NHS).
In some embodiments, the superparamagnetic particles have a size of from 50 nm to 1000 nm, preferably 100 nm.
In some embodiments, the superparamagnetic particles are functionalized to carry amino or carboxyl groups on the surface of the superparamagnetic particles to react with the carboxyl or amino groups of a biomolecule interaction pair (e.g., a nucleic acid or an antibody).
In some embodiments, the biological sample is labeled or captured by a method selected from the group consisting of substrate capture and superparamagnetic particle capture.
In some embodiments, the substrate capture method comprises reacting a biological sample by dropping the biological sample on a diamond surface containing an ensemble NV color center sensor, and then reacting by dropping superparamagnetic particles.
In some embodiments, the superparamagnetic particle capture method comprises dropwise adding superparamagnetic particles to a biological sample for reaction, and then dropwise adding to a diamond surface containing an ensemble NV centroid sensor for reaction.
In some embodiments, the superparamagnetic particle capture method further comprises placing magnets at the sides and bottom of the reaction vessel to magnetically purify and magnetically enrich the superparamagnetic particles.
In some embodiments, the method of making an ensemble diamond NV colour centre sensor is: for a block with dimensions of 2 mm × 2 mm × 0.5 mm and a crystal orientation of [100 ]]Is implanted into the electron-level purity block diamond at an energy of 15 keV14N+Ion at a dose of 2X 1013 cm-2Then annealed at 1000 deg.C for 4 hours to form a layer of high density NV color centers at a concentration of about 1.7X 10 on the near-surface of the diamond to a depth of about 20 nm11/cm2。
In some embodiments, the magnetic particles of 100nm level are used as both a tool for magnetic separation and a signal source for magnetic imaging, so that the magnetic separation of minute level can be realized, and a magnetic field with proper strength and space extension can be provided, and high-contrast magnetic images and high detection flux can be guaranteed.
In some embodiments, the magnetic particles are biologically functionalized to facilitate subsequent biomolecule recognition and single particle magnetic imaging.
In some embodiments, the target biomolecules are enriched and purified by magnetic separation, then the magnetic particle-biomolecule complex is treated by ultrasound and the like to restore single-particle dispersibility, and finally single-particle magnetic imaging detection is performed, so that the detection specificity and sensitivity are improved while the single-particle characteristics of magnetic imaging are guaranteed.
In some embodiments, a single-particle magnetic image is obtained by magnetically imaging magnetic particles on the diamond surface using a wide-field magnetic microscope with the diamond NV colour centre as a two-dimensional magnetic sensor.
In some embodiments, the single-particle magnetic field in the single-particle magnetic image is intelligently identified using a deep learning model, and is statistically analyzed for target biomolecular interactions through quantitative data.
In some embodiments, the deep learning model is a conditional challenge generative model (cGANs).
In some embodiments, the software that counts the number of magnetic particles on the surface of the diamond is ImageJ.
In some embodiments, the invention can be used for detecting protein-protein, protein-nucleic acid, nucleic acid-nucleic acid and other interactions in biomedical research, and can also be used for detecting various types of biomolecules such as protein, nucleic acid, virus and the like in clinical, customs and other scenes.
The biomolecular interaction magnetic detection method based on single-particle magnetic imaging can be widely applied to the interaction of various biomolecules, including protein-protein, protein-nucleic acid, nucleic acid-nucleic acid and the like, and provides a new technical means for relevant biomedical basic research and clinical diagnosis. In particular, the detection of a plurality of biomarkers is carried out by relying on the interaction of biological macromolecules, and the invention has great significance for the detection of various biological macromolecules in clinic and customs for diagnosis. For example, antigens or antibodies of diseases such as a new coronavirus in blood can be detected with high sensitivity by antigen-antibody interaction, thereby enabling rapid diagnosis; by the interaction between complementary DNA molecules, a proper DNA sequence can be designed to detect ctDNA in body fluid, thereby being helpful for early diagnosis of cancer.
Definition of
Magnetic detection: and (3) detecting the magnetic properties of the sample, such as the magnetic field intensity, the direction and the like.
Ensemble diamond NV colour center sensor: the nitrogen-vacancy colour centre within the diamond body is known as the NV colour centre. By surface implantation, a large number of NV colour centers are created within the diamond, which is called an ensemble diamond NV colour center sensor.
Superparamagnetic particles: the superparamagnetism means that the superparamagnetism shows paramagnetic property under the action of an external magnetic field, the direction of the generated magnetic field is consistent with that of the external magnetic field, but the magnetic susceptibility of the superparamagnetism is far higher than that of a common paramagnetic substance. Superparamagnetic particles are magnetic particles having superparamagnetism.
Biomolecule interaction pairs: a pair of interacting biomolecules, such as antigen-antibodies, can be referred to as a biomolecule interaction pair.
Deep learning model: the model for deep learning training mainly comprises a convolutional neural network model (CNN), a deep belief network model (DBN), a generation countermeasure network (GAN) and the like.
Magnetic purification: the molecules on the superparamagnetic particles react with only one kind of substances which interact with the superparamagnetic particles in a complex system and are fixed on the surfaces of the magnetic particles, but do not react with other substances in the system. Under the action of an external magnetic field, the magnetic particles can be separated from the system, so that only one kind of required substances is extracted, and the substance purification is realized. And the process performed with magnetic particles is called magnetic purification.
Magnetic enrichment: the superparamagnetic particles react with a low-concentration substance in a system and are fixed on the surfaces of the magnetic particles, the magnetic particles can be separated from the system under the action of an external magnetic field, and the low-concentration substance is also separated. And this process, which is done using magnetic particles, is called magnetic enrichment.
Single particle magnetic image: the magnetic field distribution pattern generated by the magnetic particles on the diamond surface can reflect the distribution of single magnetic particles on the diamond surface because the magnetic field distribution of the single magnetic particles is very concentrated, and the image is called a single-particle magnetic image.
Wide field magnetic imaging microscope: wide field refers to direct imaging of the field of view rather than a single point scan, as opposed to confocal. The wide-field magnetic imaging microscope is a wide-field fluorescence microscope which is modified so that the wide-field fluorescence microscope can image the magnetic field distribution.
Marking: a label (e.g., a radioisotope, a fluorescent molecule or protein, a nanoparticle, etc.) is covalently attached to the analyte such that the labeled analyte can be identified and detected by suitable means.
Capturing: by some means, a species of interest in a sample is bound to a substrate, molecule or particle.
Passivation: the term "oxidation treatment" is used to mean the treatment of a metal surface to improve its corrosion resistance, and is used herein to treat the surface of a substrate by chemical means so that it is effective against non-specific adsorption.
Single molecule interactions: that is, the interaction between only one molecule involved in the interaction, e.g., an antibody on the magnetic particle and an antigen on the diamond surface, is the interaction of a single molecule. A single molecule can be as small as several to several tens of bases of oligonucleotides or short peptides of several amino acid residues.
Advantageous effects
The invention provides a high-sensitivity and high-specificity method for biomolecule interaction detection based on magnetic measurement of diamond NV color center, and can be applied to numerous occasions. The beneficial effects of the invention are explained in detail as follows:
single molecule sensitivity: by means of NV color center magnetic imaging, single magnetic particles can be distinguished and imaged. The present invention has single molecule sensitivity because the interaction between the single molecules can ensure the magnetic particles to be bonded on the surface of the diamond.
High sensitivity: on one hand, the concentration of the biomolecule interaction pairs to be detected can be effectively improved by utilizing a magnetic enrichment means, so that the biomolecule interaction pairs with lower concentration in a sample can also generate signals which are obviously different from a control group of molecules without interaction; on the other hand, compared with a fluorescent label, the magnetic label has the characteristics of high stability, low background and high excitation efficiency, and can realize the signal detection rate close to 100 percent, thereby improving the sensitivity.
High specificity: on one hand, compared with similar schemes based on other labels, besides the specificity of characteristic recognition and combination among biomolecules and the specificity brought by passivation treatment of surface PEG, the invention can also purify the interaction pairs of the biomolecules to be detected in a sample by utilizing a magnetic purification means, thereby eliminating the interference of other biomolecules existing in the sample to a great extent and further improving the specificity; on the other hand, the magnetic background in the biological sample is low, and the read single-particle magnetic signal can be guaranteed to come from the magnetic label.
The biomolecular interaction magnetic detection method based on single-particle magnetic imaging can make up the defects of other magnetic detection means in the aspects of sensitivity and spatial resolution, and can research the biomolecular interaction on a single-molecule level. The method provided by the invention has the characteristics of small sample demand, high sensitivity, low background, high stability, high specificity and high efficiency. Therefore, the invention can provide a new technical means in the aspect of biomolecule interaction detection, and has important significance in biomolecule interaction research under complex physiological conditions, mechanism research of related diseases and clinical diagnosis and treatment. The invention provides a complete set of schemes from diamond surface biological functionalization to biological sample preparation, single-particle magnetic imaging to data processing and analysis, and is easy to popularize and use.
Drawings
Fig. 1 shows an energy level structure of a nitrogen-vacancy color center (NV color center) in diamond.
FIG. 2 shows a continuous spectrum of NV color centers.
Fig. 3 shows the distribution of superparamagnetic particles in the NV axis under an external magnetic field.
Fig. 4 shows a flow diagram according to an embodiment of the invention.
Fig. 5 shows a flow diagram of a functionalization process of a diamond surface according to an embodiment of the invention.
Fig. 6 shows a reaction and binding protocol for a biological sample according to an embodiment of the invention, wherein: a is a substrate capture method; b is a magnetic particle capture method.
FIG. 7 shows a flow chart of single particle magnetic imaging according to an embodiment of the invention.
Fig. 8 shows the use of a single-particle magnetic image to obtain a single-magnetic-particle distribution.
Fig. 9 shows an example demonstrating RBD antigen interaction with its antibodies using the present invention.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
As shown in fig. 4, the present invention captures the biomolecular interaction pair in the sample to be detected by using the functionalized diamond surface, identifies the biomolecular interaction pair by using superparamagnetic particles, performs magnetic imaging with optical resolution on the magnetic particles on the diamond surface by using a wide field magnetic microscope, identifies and counts single particles in the image by data processing, and further performs quantitative analysis on the biomolecular interaction to be detected.
The principle of the scheme is as follows:
the nitrogen-vacancy color centers (NV color centers) in diamond are used by us for magnetic field detection, and their energy level structure is shown in fig. 1. I0>Sum of states | + -1>There is a zero field cleave of 2.87 GHz between the states. NV can be excited from the ground state by a 532 nm laser. In the excited state, |0>The state relaxes directly to the ground state and radiates photons, and | + -1>In addition to the above-mentioned transition process, there is a relaxation of a part of the states to the ground state via metastable states (singlet states) by means of nonradiative transitions, which is free of photon radiation. Thus, the quantum state of NV can be read out optically. Under an off-axis magnetic field, | +/-1 of degeneracy>Zeeman splitting of the state occurs (splitting is proportional to the external magnetic field, Δ = 2 γ)eB0Wherein γ iseGyromagnetic ratio of NV). As shown in FIG. 2, in the present invention, a magnetic field is detected in a continuous wave manner, in the process of continuous laser excitation, the microwave frequency is scanned, and the fluorescence emitted from NV is collected, so that a significant fluorescence count drop occurs at the resonance position, thereby obtaining the resonance frequency, and further obtaining the magnitude of the external magnetic field according to Zeeman splitting. In the present invention, we use the ensemble diamond NV colour centre for magnetic field measurements and magnetic imaging.
Under an off-axis magnetic field, the superparamagnetic particles magnetize and thus exhibit an on-axis magnetic signal. When the magnetic field is the total magnetic field of the external magnetic field and the magnetic field of the magnetic particles, the magnetic field distribution of the magnetic particles can be obtained only by subtracting the influence of the external magnetic field. As shown in fig. 3, the signal of the superparamagnetic particles is a magnetic dipole signal. The intensity of the magnetic field is rapidly attenuated along with the extension of the distance from a signal source, the external magnetic field of the wide-field magnetic imaging microscope is a more uniform magnetic field with less change, the distribution of the external magnetic field can be better extracted through quadratic fitting of the measured magnetic field distribution, and the magnetic distribution image of the magnetic particles can be obtained by subtracting the external magnetic field. The magnetic distribution image of the magnetic particles exhibits an individual petaloid distribution, referred to herein as a single-particle magnetic image. By identifying and counting the magnetic particles in the single-particle magnetic image, the interaction of the detected biomolecules can be quantitatively analyzed.
Example 1 diamond surface functionalization treatment
As shown in fig. 5, the surface treatment of the diamond sample according to the present invention sequentially includes hydroxylation, amination, and polyethylene glycol (PEG) and Biotin (Biotin). PEG molecules can weaken electrostatic and hydrophobic interactions between the diamond surface and molecules in solution, so pegylation can passivate the diamond surface, eliminating non-specific adsorption. Meanwhile, biotin-PEG can realize the functional modification of the diamond surface, so that streptavidin can be combined to the diamond surface through the interaction of biotin-streptavidin, and any subsequent biomolecule marked with biotin can be combined to the diamond surface by taking the streptavidin as a bridge because one streptavidin is provided with four binding sites of biotin. The specific process flow of the treatment is as follows:
1. hydroxylation: adding the diamond into a piranha solution (concentrated sulfuric acid: hydrogen peroxide = 3: 1), and reacting for 2 h at 180 ℃. And taking out the diamond, washing with deionized water, and drying. This step may be optionally repeated as necessary (usually, hydroxylation can be achieved by one reaction, and the effect can be further improved by increasing the number of treatments).
2. Amination: adding diamond into aminosilane (the system is prepared according to the volume ratio of the aminosilane to acetic acid to methanol being 1: 5: 100), and reacting for 1 hour at room temperature in a dark place. And after the reaction is finished, taking out the diamond, rinsing the diamond with methanol for three times to remove unreacted aminosilane, rinsing the diamond with ultrapure water for 5 times, and drying the diamond.
3. Passivation and biotin modification: 1 mg of methylated polyethylene glycol succinimidyl valerate (mPEG-SVA, Laysan Bio. Inc., MW 5,000 or 20,000) and 9 mg of biotinylated polyethylene glycol succinimidyl valerate (Biotin-PEG-SVA, Laysan Bio. Inc., MW 5,000 or 20,000) were dissolved in 50. mu.l sodium bicarbonate solution (0.1M) (SVA is an activated carboxyl group that is very susceptible to hydrolysis in aqueous solution and thus fails, so it should be noted that the solution was ready to be formulated. Such experiments require certain preliminary experiments to determine the appropriate conditions. Different batches of diamonds have different surface conditions and different samples under different experimental conditions, so the proportion and the dosage of PEG and Biotin-PEG are required to be adjusted), the mixture is dripped on the surface of the diamond subjected to amination treatment, the mixture is put into a wetting box (1/2-3/5 volume of deionized water is filled in a waste 1 ml gun head box to preserve moisture and prevent the sample from being dried on the surface of the diamond) to react for 4 hours at room temperature or stays overnight in a refrigerator at 4 ℃, and the mixture is washed with the deionized water for 5 times and is dried. This step can be optionally repeated as necessary (usually, pegylation can be achieved by one reaction, and the effect can be further improved by increasing the number of treatments).
4. Streptavidin modification: after passivation and biotin modification, 0.6 mg/ml streptavidin (Sigma-Aldrich, S0677) solution was added dropwise to the diamond surface for 30 min, and washed 5 times with phosphate solution (1 × PBS, pH = 7.4).
5. And (optional) dropping biotinylated antibody (biotin antibody is added after streptavidin modification because the antibody is fixed on the surface of the diamond through the biotin-streptavidin interaction) on the surface of the diamond for reaction for 30 min, and washing the surface of the diamond for 5 times by using phosphate solution. This step can be omitted if the pair of biomolecular interactions to be detected contains a biotin tag.
Example 2 sample preparation
The invention identifies the interaction of biological molecules by a double sandwich mode, namely commonly called a 'three-Ming-method'. In this embodiment, in addition to the diamond functionalization, the magnetic particles also need to be functionalized accordingly. The functionalized magnetic particles of the present invention can be obtained by reacting amino groups or carboxyl groups on the surface of magnetic particles with carboxyl groups or amino groups of a biological sample (e.g., nucleic acids or antibodies) through one-step or two-step EDC/NHS crosslinking, or can be purchased directly (e.g., Ocean nanotech). The magnetic particles used in the invention are generally 100 nm-sized by comprehensively considering factors such as signal intensity and distribution range, magnetic separation efficiency, steric hindrance and the like of the magnetic particles under a certain external magnetic field.
The EDC/NHS cross-linking step in one or two steps is the same as that used to treat the magnetic particles in other applications, either according to literature procedures or as specified by the purchasing company. The detailed steps are as follows:
a one-step method:
1. 0.25 mL of magnetic particles (10 mg/mL) were removed in a 1.5 mL Ep tube, which was placed on a magnetic separator.
2. The supernatant was removed. The magnetic particles were redispersed by adding 0.4 ml activation buffer (25 mM MES (4-morpholinoethanesulfonic acid), pH = 6.0).
3. Mu.l EDC solution (10 mg/mL) was added (EDC being the carboxyl and amine reactive zero length crosslinker).
4. The reaction was carried out for 15 minutes at room temperature with continued mixing. To the beads was added 0.25 ml of protein (often an antibody to the captured biomolecular interaction pair, such as the interaction of A and B to be detected. if an antibody to A is on the diamond, an antibody to B is modified to the beads) (1 mg/ml).
5. The reaction was continued for 2.5 hours at room temperature with continued mixing.
6. The reacted tube was placed on a magnetic separator and separated for 5 minutes to completely separate the magnetic particles (the specific magnetic separation time depends on the magnetic field of the magnetic separator).
7. The supernatant was removed and PBST buffer was added. The magnetic particles are re-dispersed by oscillation and ultrasound.
8. Repeat step 6-7 three times. Finally, the magnetic particles are re-dispersed in PBST, the volume is fixed to 1 mg/mL of the magnetic particle concentration, and the magnetic particles are stored in a refrigerator at 4 ℃.
A two-step method:
1. 0.25 mL of magnetic particles (10 mg/mL) were removed in a 1.5 mL Ep tube, which was placed on a magnetic separator.
2. The supernatant was removed. The magnetic particles were redispersed by adding 0.4 ml activation buffer (25 mM MES, pH = 6.0).
3. 12.5. mu.l of Sulfo-NHS solution (10 mg/mL) and 4.5. mu.l of EDC solution (10 mg/mL) were added.
4. The reaction was carried out for 15 minutes at room temperature with continued mixing.
5. The supernatant was removed by magnetic separation, and then the magnetic particles were redispersed in an activation buffer by sonication.
6. To the beads was added 0.25 ml of protein (often an antibody to the captured biomolecular interaction pair, such as the interaction of A and B to be detected. if an antibody to A is on the diamond, an antibody to B is modified to the beads) (1 mg/ml).
5. The reaction was continued with mixing at room temperature for 2.5 hours.
6. The reacted tube was placed on a magnetic separator and separated for 5 minutes to completely separate the magnetic particles (the specific magnetic separation time depends on the magnetic field of the magnetic separator).
7. The supernatant was removed and PBST buffer was added. The magnetic particles are redispersed by oscillation and ultrasound.
8. Repeat step 6-7 three times. Finally, the magnetic particles are re-dispersed in PBST, the volume is determined to be 1 mg/mL of magnetic particle concentration, and the magnetic particles are stored in a refrigerator at 4 ℃.
As shown in fig. 6, the present invention provides two solutions to achieve a "sandwich" of diamond surfaces: one solution is to mix functionalized magnetic particles with the sample, so that the interaction pairs to be detected in the sample are captured by the magnetic particles and then react with the diamond surface. Referred to herein as magnetic particle capture. By utilizing the characteristic of magnetic separation of magnetic particles in an external magnetic field, a system formed by the reaction of the magnetic particles and a sample to be detected can be selected for magnetic purification and magnetic enrichment so as to improve the sensitivity and specificity. Another solution is to react the sample with the functionalized diamond surface first, so that the interaction pair to be detected is captured on the diamond surface first, and then the interaction pair on the diamond surface by the functionalized magnetic particles is labeled, which is referred to herein as substrate capture method. The specific reaction flow is as follows, and any scheme can be selected according to the purpose and the requirement of the experiment:
a) substrate capture method:
1. and dropwise adding a sample to be detected on the surface of the diamond (in order to obtain single particle distribution, proper biological sample concentration is used, and the proper sample concentration is adjusted according to the actual density of surface biomolecule interaction pairs, wherein due to the too high concentration, surface molecules are too dense, unimolecules are difficult to distinguish and statistics are performed, and the sample with too low concentration has too low bonding efficiency on the surface of the diamond, so that a signal can not be detected almost), and reacting for 1 h at room temperature. Washed 5 times with phosphate buffered saline (1 × PBS).
2. 0.02 mg/ml of the corresponding antibody or nucleic acid-modified magnetic beads was added dropwise thereto, followed by reaction for 30 min and washing with phosphate buffer (1 XPBS) for 5 times. And (4) quickly changing and washing the diamond by ultrapure water once, and drying the diamond by blowing so as to carry out single-particle magnetic imaging.
b) Magnetic particle capture method:
1. and (3) dropwise adding an appropriate amount of (generally 1/5-1/50 of the amount of the interactive molecular substances to be captured in the sample to be detected according to the volume and the concentration of the sample to be detected so as to ensure that enough magnetic particles can be enriched and capture is arranged on each magnetic particle) magnetic beads into the sample to be detected, fully mixing, and reacting for 30 min.
2. (optional) magnetic purification and magnetic enrichment: the magnet was placed on the side and bottom of the reaction vessel for about 5-10 min, after the magnetic particles were completely adsorbed near the magnet, the supernatant was aspirated as much as possible, then 100. mu.l of PBST buffer (1 XPBS, 0.01% Tween20, pH = 7.4) was added, shaken well, and the above procedure was repeated 5 times. The supernatant was aspirated for the last time and the volume was adjusted to 5-100. mu.l. And (3) carrying out ultrasonic treatment on the sample with constant volume for 5 min to ensure that the magnetic particles are fully dispersed, and prolonging the time according to the experimental requirement. Then, the reaction was left for 30 min to ensure that the biomolecules partially detached by the ultrasound were re-bound to the magnetic particles.
3. And (3) adding the sample reacted in the step (1) or the step (2) to the surface of the diamond through reasonable dilution and dropwise reacting for 1 h, and washing for 5 times by using a phosphate buffer solution. And (4) quickly changing and washing the diamond by ultrapure water once, and drying the diamond by blowing so as to carry out single-particle magnetic imaging.
Example 3 Single particle magnetic imaging
After the biomolecular interaction pairs and the magnetic particles are fixed on the surface of the diamond, the magnetic particles on the surface of the diamond need to be magnetically imaged on a wide-field magnetic imaging microscope by using a two-dimensional quantum magnetic sensor (the two-dimensional quantum magnetic sensor is one of the aforementioned ensemble diamond NV sensors, and NV color centers are distributed in a single layer in the whole diamond surface), and then statistics and analysis are carried out.
The preparation process of the two-dimensional quantum magnetic sensor comprises the following steps: for a block with dimensions of 2 mm × 2 mm × 0.5 mm and a crystal orientation of [100 ]]The electron-level purity of the bulk diamond (Element Six) is implanted at an energy of 15 keV14N+Ion at a dose of 2X 1013 cm-2(this energy and dose are both achieved by an ion implanter) and then annealed at 1000 ℃ for 4 hours to form a layer of high density NV color centers at a concentration of about 1.7X 10 on the near-surface of the diamond to a depth of about 20 nm11/cm2. This ensures high sensitivity and submicron resolution of the two-dimensional magnetic quantum sensor used in the present invention, thereby ensuring detection efficiency and detection flux.
The measurement flow is shown in fig. 7. The invention uses a wide field magnetic imaging microscope device (CN 114113151A) to obtain single particle magnetic images. Since the external magnetic field can cause zeeman splitting of NV level to change its resonant frequency, the magnetic field can be detected by scanning the microwave to obtain the change of its resonant frequency, so that a single-particle magnetic image can be obtained by continuous wave spectrum (CW) detection, and the specific process is explained as follows:
1. and fixing the sample stage on a base of the objective lens, and focusing the sample.
2. The front end optical path is adjusted such that the optical path achieves total internal reflection.
3. The computer controls the microwave output to fix the frequency, and controls the camera to collect a fluorescence image (collecting a fluorescence image of an NV color center in the diamond).
4. Changing the microwave frequency, repeating the step 3, scanning a plurality of frequency points near the resonance frequency, and acquiring a fluorescence image.
5. Repeating steps 2 and 3 for a plurality of times to obtain a magnetic image with high signal-to-noise ratio and high contrast. The number of repetitions is related to the nature of the NV colour centre of the diamond sample. The better the NV color center property, the higher the sensitivity of magnetic measurement, the shorter the time required for obtaining a magnetic image with high signal-to-noise ratio and high contrast, and the fewer the repetition times. The sensitivity of measuring magnetism refers to the change of a magnetic field measured in unit time, and the sensitivity of measuring magnetism by continuous waves is
Wherein ΔfIt is referred to as the full width at half maximum,Cit is referred to as the contrast ratio,Rrefers to the photon count rate. The better the NV color center property, the higher the contrast, the narrower the full width at half maximum, and the higher the corresponding sensitivity of magnetic measurement, the shorter the time required for measuring the same magnetic field.
6. The corresponding fluorescence images collected at each frequency were averaged.
7. The continuous spectrum of each pixel in the post-fluorescence image is averaged using Lorentzian peak fitting (fitting the parameters of the Lorentzian peak by L-M (Levenberg-Marquarelt) algorithm to minimize error from experimental data. other means of non-linear fitting within matlab origin can also be used) and the resonance frequency profile is obtained and converted to a magnetic profile.
8. And performing second-order polynomial fitting on the magnetic field on the surface of the diamond, and deducting the fitting graph from the original graph to obtain a magnetic signal distribution graph.
9. (optional) obtaining single particle magnetic images of a plurality of different regions on the diamond as desired.
Example 4 data processing and analysis
The original magnetic field image appears to be chaotic in signal because the magnetic field exhibits north and south polarities. In order to visually acquire the quantity information of the magnetic particles on the surface of the diamond, the invention uses a deep learning model (T. -C. Wang et al., in Proceedings of the IEEE conference on computer vision and pattern recognition) (2018), pp. 8798-. For the image reconstructed by inverse solution, the number of single particles in the image can be rapidly counted in batches by using common software such as ImageJ and the like, for example, the number of magnetic signals in the same area is different under the interaction of different intensities under the same concentration, the interaction intensities among different molecules can be compared by counting the density of the magnetic signals, and the interaction of target biomolecules is analyzed by quantitative data, so that the research of the biological function and the diagnosis of clinical medicine of the target biomolecules are guided.
Example 5 analysis of the RBD antigen-antibody interaction of New coronavirus
The invention is further illustrated below with reference to the novel coronavirus RBD antigen-antibody interaction as an example.
Sensor for obtaining NV color center of heald diamond
Two pieces with the size of 2 mm multiplied by 0.5 mm and the crystal orientation of 100]The same batch of electron-level purity bulk diamond (Element Six) is placed in an ion implanter and implanted with energy of 15 keV14N+Ion at a dose of 2X 1013 cm-2And then taken out to be annealed at 1000 ℃ for 4 hours. Forming a high density layer at a depth of about 20 nm from the surface of the diamondDegree NV color center at a concentration of about 1.7X 1011/cm2。
Surface functionalization of diamond
Two diamonds were treated in the same way.
1. Hydroxylation: diamond was added to a piranha solution (concentrated sulfuric acid: hydrogen peroxide =6 ml: 2 ml) and reacted at 180 ℃ for 2 h. And taking out the diamond, washing with deionized water, and drying. The above steps are repeated once.
2. Amination: diamond was added to aminosilane (in a ratio of 50. mu.l aminosilane, 5 ml methanol, 250. mu.l acetic acid) and reacted for 1 hour at room temperature with exclusion of light. After the reaction is finished, the diamond is taken out, rinsed for three times by methanol to remove unreacted aminosilane, rinsed for 5 times by ultrapure water and dried.
3. Passivation and biotin modification: 5 mg of biotinylated polyethylene glycol succinimidyl valerate (Biotin-PEG-SVA, Laysan Bio. Inc., MW 20,000) was dissolved in 50. mu.l sodium bicarbonate solution (0.1M, pH = 8.3), added dropwise to the aminated diamond surface, rinsed 5 times with deionized water and blown dry overnight in a humidified box at 4 ℃ in a refrigerator. The above steps are repeated once.
4. Streptavidin modification: after passivation and biotin modification were complete, a 2. mu.l solution of streptavidin (0.6 mg/ml, Sigma-Aldrich, S0677) was added dropwise to the diamond surface for 30 min and washed 5 times with phosphate solution (1 XPBS, pH = 7.4).
Magnetic particle marking
1. 0.02 mL of magnetic particles (10 mg/mL, Ocean nanotech, SC0100, 100 nm) were removed in a 1.5 mL Ep tube, which was placed on a magnetic separator.
2. The supernatant was removed. The magnetic particles were redispersed by adding 0.1 ml activation buffer (25 mM MES, pH = 6.0).
3. Mu.l of Sulfo-NHS solution (10 mg/mL) and 10 mu.l of EDC solution (10 mg/mL) were added.
4. The reaction was carried out for 15 minutes at room temperature with continued mixing.
5. The supernatant was removed by magnetic separation, and then the magnetic particles were redispersed in an activation buffer by sonication.
6. To the beads, 0.02 ml of goat anti-rabbit antibody (2.3 mg/ml, Jackson ImmunoResearch, 111-.
5. Mixing was continued overnight at 4 ℃.
6. The reacted tube was placed on a magnetic separator and separated for 5 minutes to completely separate the magnetic particles.
7. The supernatant was removed and PBST buffer was added. The magnetic particles are redispersed by oscillation and ultrasound.
8. Repeat steps 6-7 five times. Finally, the magnetic particles are re-dispersed in PBST, the volume is fixed to 1 mg/mL of the magnetic particle concentration, and the magnetic particles are stored in a refrigerator at 4 ℃.
Sample preparation
1. Mu.l of biotinylated RBD (0.1 mg/ml, Nano Biological, 40592-V08B-B) was added dropwise to both diamond surfaces for 30 min, and washed 5 times with phosphate solution.
2. The experimental diamond was added with 2. mu.l rabbit-derived RBD antibody (100 ng/ml, Genetex, GTX 635792) dropwise for 30 min, and washed 5 times with phosphate solution. The control diamond was stored in phosphate buffer.
3. Two diamonds were added 0.02 mg/ml goat anti-rabbit antibody-modified magnetic beads dropwise for 15 min, and washed 5 times with phosphate buffered saline (1 XPBS). Quickly changing and washing once with ultrapure water, and drying.
Single particle magnetic imaging
The same operation is carried out on two diamonds
1. The diamond was fixed on a sample stage fixed to the base of the objective lens and focused on the diamond surface with a 100-fold air mirror (Olympus, MPLFLN 100 ×, Numerical Aperture (NA) 0.9).
2. The front end optical path is adjusted such that the optical path achieves total internal reflection. The diamond NV colour centre is excited by a 532 nm laser.
3. The computer controls the microwave output to fix the frequency, and simultaneously controls the camera to acquire a fluorescence image generated by the NV color center of the diamond within 0.1 s of exposure time
4. Changing the microwave frequency, repeating the step 3, scanning a plurality of frequency points near the resonance frequency, and acquiring a fluorescence image.
5. Repeating the step 2 and the step 350 times to obtain the magnetic image with high signal-to-noise ratio and high contrast ratio.
6. The corresponding fluorescence images collected at each frequency were averaged. Thus, a group of fluorescence maps which change along with the microwave frequency is obtained, and the change of the fluorescence intensity of each pixel point along with the frequency is the continuous wave spectrum of each pixel.
7. Fitting Lorentz peak to continuous spectrum corresponding to each pixel in the averaged fluorescence image by L-M (Levenberg-Marquarelt) algorithm to obtain resonance frequency distribution diagram, and obtaining the resonance frequency distribution diagram according to Zeeman splitting frequency delta = gammae B . (γe= 2.80 MHz/gauss) to convert the resonance frequency map into magnetic distribution maps.
8. And performing second-order polynomial fitting on the magnetic field on the surface of the diamond to obtain an external magnetic field distribution graph, and deducting the fitting graph from the original graph to obtain a single-particle magnetic image.
Data processing and analysis
1. Simulating the magnetic field distribution of the diamond surface according to a dipole field by signals generated by magnetic particles with certain distribution, and generating a deep learning model of a countermeasure network by using a simulated image pair condition for training, wherein the method comprises the steps of acquiring real magnetic moment data; obtaining magnetic field data corresponding to the real magnetic moment data according to the real magnetic moment data; acquiring a training sample set according to the magnetic field data and the real magnetic moment data; training the magnetic field reconstruction model to be trained by utilizing the training sample set to obtain a trained magnetic field reconstruction model; and wherein training the magnetic field reconstruction model to be trained using the training sample set comprises: the magnetic field data input condition is used for generating a generator of the countermeasure network so that the generator generates simulated magnetic moment data, and the real magnetic moment data and the simulated magnetic moment data input condition are used for generating a discriminator of the countermeasure network so that the discriminator discriminates the truth of the simulated magnetic moment data.
2. As shown in fig. 8, the single-particle magnetic image is guided into the trained neural network to perform inverse solution to reconstruct a single-particle distribution image.
3. The number of magnetic signals of the inversely reconstructed image was counted using ImageJ.
As shown in fig. 9, this embodiment demonstrates most of the advantageous effects of the present invention. The signal in the image of the invention is a single-particle signal, and the single-molecule sensitivity is realized because the single magnetic particle can be fixed on the surface of the diamond through the interaction between single molecules. The signal in the invention has no background and good stability, and can ensure nearly 100% readout. The control group of the present invention showed almost no signal, demonstrating the passivation of surface PEG and the high specificity of the magnetic label.
This example demonstrates that there is a strong interaction between the RBD and its antibody, and little interaction between the secondary antibody. Utilize to have stronger interact between RBD and its antibody, can adopt this scheme to detect the antibody that whether contains new coronavirus in the serum, whether infect novel coronavirus to everybody in a large amount of crowds through serum epidemiology carries out quick diagnosis, judges the infection risk that exists among the crowd and tracks the infector that probably exists to block the propagation of disease.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for magnetic detection of biomolecular interactions, comprising the steps of:
a. labeling or capturing a biomolecular interaction pair in a biological sample by a superparamagnetic particle, thereby binding the biomolecular interaction pair on a functionalized diamond surface comprising an ensemble NV color center sensor;
b. magnetically imaging the superparamagnetic particles on the surface of the diamond containing the ensemble NV color center sensor by using a wide-field magnetic imaging microscope;
c. and establishing a mapping relation between the obtained magnetic image and the distribution of the superparamagnetic particles through a deep learning model, performing inverse solution and reconstruction on the distribution of the superparamagnetic particles, then counting the number of the superparamagnetic particles on the surface of the diamond, and analyzing the interaction of the target biomolecules through quantitative data of the superparamagnetic particles.
2. The method of claim 1, wherein the functionalization of the diamond surface containing ensemble NV colour centre sensors is achieved by biotin/streptavidin engineering.
3. The method of claim 1, wherein functionalizing the diamond surface containing the ensemble NV colour center sensor further comprises passivating the diamond surface by pegylation modification.
4. The method of claim 1, wherein the functionalization of the diamond surface containing ensemble NV colour centre sensors comprises hydroxylation, amination, passivation and biotin and streptavidin modification of the diamond surface containing ensemble NV colour centre sensors, optionally further comprising treatment of the diamond surface containing ensemble NV colour centre sensors with biotinylated antibodies.
5. The method according to claim 4, characterized in that the hydroxylation is carried out by means of a piranha solution and the amination is carried out by means of an aminosilane.
6. The method according to claim 3 or 4, wherein the passivation is performed by methylating polyethylene glycol succinimidyl valerate or methoxypolyethylene glycol active ester.
7. The method of claim 4, wherein the biotin modification is performed by biotinylating polyethylene glycol succinimidyl valerate or biotinylating polyethylene glycol active ester.
8. The method of claim 1, wherein the superparamagnetic particles are functionalized to carry amino or carboxyl groups on their surface for reaction with carboxyl or amino groups of a biomolecular interaction pair.
9. The method of claim 1, wherein the biological sample is labeled or captured by a method selected from the group consisting of substrate capture and superparamagnetic particle capture, wherein the substrate capture comprises dropping the biological sample on a diamond surface containing an ensemble NV centroid sensor for reaction, and then dropping superparamagnetic particles for reaction; the superparamagnetic particle capture method comprises the steps of dropwise adding superparamagnetic particles into a biological sample for reaction, and then dropwise adding the superparamagnetic particles onto the diamond surface containing the ensemble NV color center sensor for reaction.
10. The method of claim 1, wherein the superparamagnetic particle capturing method further comprises placing magnets at the side and bottom of the reaction vessel to magnetically purify and enrich the superparamagnetic particles.
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