METHOD FOR DETECTING MICROPROTEINS IN SAMPLE SYSTEM

Abstract

A method for detecting a trace protein in a sample system, comprising: providing a primary antibody-modified immunomagnetic bead and a secondary antibody-modified nanoparticle for a to-be-detected protein; mixing and incubating the primary antibody-modified immunomagnetic bead and the secondary antibody-modified nanoparticle with a sample system containing the to-be-detected protein, so as to form a complex having a double-antibody sandwich structure; and allowing the complex to pass through a micro-nano pore device in the form of a single particle and trigger an electrical pulse signal, analyzing the electrical pulse signal to obtain a charge state or a volume/mass state of the complex, and calculating, according to the charge state or the volume/mass state, the amount of the to-be-detected protein in the sample system.

Claims

1. A method for detecting a trace protein in a sample system, the method comprising: providing a primary antibody-modified immunomagnetic bead and a secondary antibody-modified nanoparticle for a to-be-detected protein; mixing and incubating the primary antibody-modified immunomagnetic bead and the secondary antibody-modified nanoparticle with a sample system containing the to-be-detected protein, so as to form a complex having a double-antibody sandwich structure; and allowing the complex to pass through a micro-nano pore device in the form of a single particle and trigger an electrical pulse signal, analyzing the electrical pulse signal to obtain a charge state or a volume/mass state of the complex, and calculating, according to the charge state or the volume/mass state, the amount of the to-be-detected protein in the sample system; wherein the electrical pulse signal reflects a charge state of the complex: the state and/or number of the nanoparticle bound on the complex is obtained by analyzing a peak fluctuation of the electrical pulse signal, and the amount of the to-be-detected protein in the sample system is calculated according to the state and/or number and a reaction principle of the double-antibody sandwich structure; wherein the volume/mass state is positively related to an integral area of the electrical pulse signal(s), the number of the protein molecules captured on the complex is directly proportional to an increase in volume/mass of the complex relative to the immunomagnetic bead, the number of the protein molecules captured on the complex is calculated according to a standard curve relationship of the integral area of the electrical pulse signal(s) and the number of the protein molecules captured on the complex, and in turn the amount of the to-be-detected protein in the sample system is calculated.

2. The detection method according to claim 1, wherein the method further comprises: in a solution system that is suitable for an antibody modification reaction, allowing the primary antibody to covalently bind to a function group on a surface of the immunomagnetic bead to obtain the primary antibody-modified immunomagnetic bead.

3. The detection method according to claim 2, wherein the degree of modification on the surface of the immunomagnetic bead by the primary antibody is adjusted by adjusting a concentration ratio of the primary antibody to the immunomagnetic bead in the solution system.

4. The detection method according to claim 1, wherein the immunomagnetic bead has a particle size of at least 1 to 1000 times that of the nanoparticle.

5. The detection method according to claim 1, wherein the micro-nano pore device is a micro-nano pore single-particle counting device based on the Coulter principle; the device comprises two cavities filled with an electrolyte and a micro-nano pore that communicates the two electrolyte-filled cavities; when the complex passes through the micro-nano pore in the form of a single particle, it transiently blocks the flowing of ions through the micro-nano pore, thereby forming an electrical pulse signal.

6. (canceled)

7. The detection method according to claim 1, wherein the micro-nano pore device is a device having a micropore-bearing double-layer film structure, the device comprises two layers of micropore-bearing nano films spaced apart by a set distance, the micropores on the two nano films are disposed opposite to each other and communicate the cavities at non-opposing sides of the nano films, and the cavities are filled with an electrolyte and are respectively provided with an electrode for maintaining ion transport; when the complex passes through the two micropores successively in the form of a single particle, a pair of electrical pulse signals having a time interval are generated.

8. The detection method according to claim 7, wherein the method further comprises: adjusting a concentration of the complex in the sample such that the electrical pulse signal interval between respective complexes is much larger than the time interval between the pair of electrical pulse signals, and analyzing a pulse intensity threshold of the electrical pulse signals so as to filter off the signals generated by immunomagnetic beads not bound with the nanoparticle.

9. The detection method according to claim 8, wherein with respect to the electrical pulse signal of the complex, an electromobility is calculated to obtain a particle surface potential of the complex. $μ = \frac{v (x)}{E (x)} = \frac{\partial v (x)}{\partial E (x)}$ $μ = \frac{ϵξ}{η}$ $ξ = A \frac{\partial (\frac{1}{r})}{\partial V}$

10. (canceled)

11. The detection method according to claim 4, wherein, the immunomagnetic bead has a particle size of generally in the range of from 100 nm to 10 μm, and the nanoparticle has a particle size of generally smaller than 1 μm.

12. The detection method according to claim 9, wherein with respect to the electrical pulse signal of the complex, an electromobility is calculated to obtain a particle surface potential of the complex; preferably, the electromobility is calculated according to the following formula: $μ = \frac{v (x)}{E (x)} = \frac{\partial v (x)}{\partial E (x)}$ where μ represents the electromobility, ν(x) represents a speed at which a complex particle passes through the two micropores successively, and E(x) represents an electric field distribution; the particle surface potential ξ corresponds to the electromobility μ in the following relationship: $μ = \frac{ϵξ}{η}$ where η represents a viscosity of the electrolyte; and, the formula for the particle surface potential ξ is as follows: $ξ = A \frac{\partial (\frac{1}{t})}{\partial V}$ where t represents a residence time of the particle in the micropores, A represents a correction factor for each micropore, and V represents a potential difference.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1 is a schematic depiction of the principle of a double-antibody sandwich structure for a single-protein complex in embodiments of the present application;

[0031] FIG. 2 is a schematic depiction of the principle of a double-antibody sandwich structure for a multiple-protein complex in embodiments of the present application;

[0032] FIG. 3 is a schematic depiction of a micro-nano pore single-particle counting device based on the Coulter principle in embodiments of the present application;

[0033] FIG. 4. is a schematic depiction of the principle of determining a charge state of a complex particle by analyzing a minute fluctuation of an electrical pulse signal peak in embodiments of the present application;

[0034] FIG. 5 is a schematic depiction of a device having a micropore-bearing double-layer film structure in embodiments of the present application;

[0035] FIG. 6 is a schematic depiction of the principle of integrally forming the device having a double-layer film structure by means of micro-nano processing in embodiments of the present application;

[0036] FIG. 7 is a schematic depiction of a pair of electrical pulse signals generated when respective complexes pass through two micropores successively in the form of a single particle in embodiments of the present application;

[0037] FIG. 8 is a pulse diagram of true electrical pulse signal pairs when respective complexes pass through two micropores successively in the form of a single particle in embodiments of the present application;

[0038] FIG. 9 is a diagram showing a linear relationship of the number of captured protein molecules v.s. the electromobility (migration speed) in embodiments of the present application; and

[0039] FIG. 10 is a diagram showing electrical pulse signals (A) and a diagram showing a standard curve relationship of the integral area of the electrical pulse signals (the volume/mass of respective complexes) v.s. the number of captured protein molecules on the complexes.

DESCRIPTION OF EMBODIMENTS

[0040] The present application will be further illustrated below by detailed description of embodiments with reference to the accompanying drawings. In the following embodiments, many details are described so that the present application will be better understood. However, those skilled in the art can readily recognize that some of the features may be omitted, or replaced by other materials or methods, depending on different situations.

[0041] Additionally, the characteristics, operations or features described in the specification can be combined in any suitable manner to form various embodiments. Moreover, the steps or actions in the description of the method may also be switched or adjusted in sequence in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the description and the drawings are merely for the purpose of clearly describing a particular embodiment and are not intended to be required, unless it is otherwise specified that a specific sequence must be followed.

[0042] The present application provides a method for detecting a trace protein in a sample system.

[0043] In embodiments of the present application, the sample system can be a simple system containing a single protein component, or can be a complex system containing one or more proteins and at the same time containing other components, including blood, plasma, serum, tissue fluid, urine, cerebrospinal fluid, among other samples.

[0044] The method of the present application enables detection of a trace protein in a sample, for examples a protein present on a microgram, nanogram or picogram scale. The method has a minimum detection limit of down to a single protein molecule, can detect a trace protein at an amount below a lower limit of detection of conventional immunological detections, and can be widely used in immunological detection, microorganism detection, and cell separation, among other fields.

[0045] The method for detecting a trace protein in a sample system according to the present application comprises the following steps:

[0046] providing a primary antibody-modified immunomagnetic bead and a secondary antibody-modified nanoparticle for a to-be-detected protein;

[0047] mixing and incubating the primary antibody-modified immunomagnetic bead and the secondary antibody-modified nanoparticle with a sample system containing the to-be-detected protein, so as to form a complex having a double-antibody sandwich structure; and

[0048] allowing the complex to pass through a micro-nano pore device in the form of a single particle and trigger an electrical pulse signal, analyzing the electrical pulse signal to obtain a charge state or a volume/mass state of the complex, and calculating, according to the charge state or the volume/mass state, the amount of the to-be-detected protein in the sample system.

[0049] In embodiments of the present application, the primary antibody and the secondary antibody respectively bind to different antigenic determinants on the to-be-detected protein to form a double-antibody sandwich structure. As shown in FIG. 1, the Y-shaped structure represents an antibody, the larger sphere represents an immunomagnetic bead modified by a primary antibody on a surface thereof, the smaller sphere represents a nanoparticle modified by a secondary antibody on a surface thereof, the rhombus structure represents a to-be-detected protein, and the formed complex having a double-antibody sandwich structure can be structurally represented as immunomagnetic bead-primary antibody-to-be-detected protein-secondary antibody-nanoparticle. The complex having a double-antibody sandwich structure shown in FIG. 1 has only one to-be-detected protein (the rhombus structure in the figure), and thus can be termed as a “single-protein complex”. The double-antibody sandwich structure according to the present application facilitates increasing capture specificity.

[0050] In embodiments of the present application, the primary antibody and the secondary antibody are not particularly limited in terms of the source and the type thereof, and can for example be a polyclonal antibody or a recombinantly expressed monoclonal antibody from various animal sources, such as an IgG antibody from a mouse, a goat, a rabbit, a horse, etc. In an embodiment of the present application, the primary antibody is mouse IgG, and the second antibody is a goat anti-mouse antibody.

[0051] In embodiments of the present application, a number of methods are available to modify the immunomagnetic bead with the primary antibody, which are not particularly limited in the present application. In a preferred embodiment, in a solution system that is suitable for an antibody modification reaction, the primary antibody is allowed to covalently bind to a function group on a surface of the immunomagnetic bead to obtain the primary antibody-modified immunomagnetic bead. The functional group on the surface of the immunomagnetic bead can be an amino group, a carboxyl group, etc. and such an immunomagnetic bead can be termed as an amino magnetic bead, a carboxy magnetic bead, etc. The surface of the immunomagnetic bead is modified by the primary antibody by means of formation of covalent binding of the function group on the surface of the immunomagnetic bead with a corresponding group on the primary antibody. A typical reaction for modifying the immunomagnetic bead with the primary antibody is achieved under the action of EDC (1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). For example, in a preferred embodiment, the immunomagnetic bead (Ademtech) is exchanged with an MES buffer (2-(N-morpholinoethanesulfonic acid) for three times, and is equilibrated until the solution has a pH of 4-5 and an ionic strength of 0.1M. Into the equilibrated solution are added 1 mg of EDC and 1 mg of NHS, each dissolved in DMSO, and reaction is allowed for 20 minutes. Then the reaction mixture is centrifuged, and the supernatant is discarded. The precipitate obtaining after discarding the supernatant is redissolved using the MES buffer, and 0.1 mg of mouse IgG (Cat.: ab151276, Abcam) as the primary antibody is added and mixed to allow coating for 1 hour. Then BSA (Cat.: 10735108001, Roche) as a terminating agent is added to a BSA concentration of 1% to terminate the reaction.

[0052] In preferred embodiments, the degree of modification of the surface of the immunomagnetic bead by the primary antibody is adjusted by adjusting a concentration ratio of the primary antibody to the immunomagnetic bead in the solution system. Single modification and multiple modification can be performed to adjust the number of secondary antibody-modified nanoparticles that can be bound, thereby forming a special complex. As shown in FIG. 2, the complex having a double-antibody sandwich structure comprises a plurality of to-be-detected protein molecules (represented by rhombus structures in the figure), and thus can also be termed as a “multiple-protein complex”. For example, in a preferred embodiment, the concentration of the primary antibody is lower than 1 μg/mg immunomagnetic bead, such that the degree of modification of the surface of the immunomagnetic bead by the primary antibody is lower than 50 antibodies/immunomagnetic bead; in another preferred embodiment, the concentration of the primary antibody is 1-5 μg/mg immunomagnetic bead, such that the degree of modification of the surface of the immunomagnetic bead by the primary antibody is lower than 300 antibodies/immunomagnetic bead; and in yet another preferred embodiment, the concentration of the primary antibody is higher than 50 μg/mg immunomagnetic bead, such that the primary antibody is saturated on the immunomagnetic bead. In this way, the amount of primary antibody modification on the immunomagnetic bead can be controlled. The degree of modification can be further confirmed by electron microscopy.

[0053] In embodiments of the present application, the nanoparticle can be selected from nanoparticles of various materials and having various suitable particle sizes and various charge states, such as a polyethylene nano microsphere, a nano magnetic bead, a nano silicon sphere, a nano latex, etc. The particle size of the nanoparticle can for example be generally smaller than 1 micrometer, and in general, the corresponding immunomagnetic bead has a particle size that is at least greater than that of the nanoparticle, which enables achieving a good performance and facilitates formation of a special complex structure.

[0054] In embodiments of the present application, the principle and the method for preparing the secondary antibody-modified nanoparticle are similar to those for the primary antibody-modified immunomagnetic bead. A number of methods are available to modify the nanoparticle with the secondary antibody, which are not particularly limited in the present application. In a preferred embodiment, in a solution system that is suitable for an antibody modification reaction, the secondary antibody is allowed to covalently bind to a function group on a surface of the nanoparticle to obtain the secondary antibody-modified nanoparticle. As the nanoparticle carries a certain amount of charge, the number of the nanoparticle in the complex having a double-antibody sandwich structure formed according to the present application will influence the charge state of the entire complex. In the present application, it is by analyzing the charge state of the complex that the number of the nanoparticle on a single complex is obtained, and in turn the amount of the to-be-detected protein is analyzed and obtained.

[0055] In a typical embodiment of the present application, a reaction for modifying the nanoparticle with the secondary antibody is achieved under the action of EDC and NHS. For example, in a preferred embodiment, the secondary antibody is a goat anti-mouse antibody, and the preparation method for the nanoparticle (Cat.: F122270, aladdin) labelled with the goat anti-mouse antibody comprises the following steps: equilibrating 1 mg of the nanoparticle in a PBS solution having a pH of 8.0 and a concentration of 0.1 M to a final concentration of the nanoparticle of 0.1 mg/mL; then adding EDC and NHS until a final concentration of each of EDC and NHS in the solution of 0.2 mg/mL, allowing activation for 30 minutes, centrifuging the reaction mixture and discarding the supernatant, redissoving the precipitate obtained after discarding the supernatant in a PBS solution, adding a goat anti-mouse antibody (Cat.: 4T20, HyteSt) and allowing labelling for 25 minutes, and adding BSA until a concentration of 0.5% to terminate the reaction.

[0056] The protein detection method according to the present application is performed based on an electrical pulse signal, which is triggered when the complex having a double-antibody sandwich structure formed during the immunological reaction undergoes electrophoresis in the electrolyte and passes through the micropore(s). A charge state of the complex can be obtained by analyzing the electrical pulse signal, and the amount of the to-be-detected protein in the sample system can be calculated according to the charge state. In an embodiment of the present application, the complex having a double-antibody sandwich structure passes through a micro-nano pore device in the form of a single particle and triggers an electrical pulse signal, which can be analyzed to obtain a charge state of the complex, which in turn can be used to calculate the amount of the to-be-detection protein in the sample system.

[0057] A number of methods are available to implement the protein detection method based on an electrical pulse signal according to the present application. One method involves a micro-nano pore single-particle counting device based on the Coulter principle (for example, U.S. Pat. No. 2,656,508, 1953), which detects signals of respective complexes in the micro-nano pore detection device. As shown in FIG. 3, the micro-nano pore single-particle counting device comprises two electrolyte-filled cavities, that is, cavity 31 and cavity 32 in the figure, and a sole micro-nano pore 33 that communicates the two electrolyte-filled cavities. When the complex passes through the micro-nano pore 33 in the form of a single particle under a driving force (such as, an electric field, a pressure, an electroosmotic flow, etc.), it transiently blocks the flowing of ions through the micro-nano pore 33, thereby forming an electrical pulse signal. The electrical pulse signal can reflect ion size, binding state, charge state and even geometry. The binding state (whether bound or not) of the nanoparticle and the binding number of the nanoparticle in the complex is obtained by analyzing a peak fluctuation of the electrical pulse signal, and the amount of the to-be-detected protein in the sample system is calculated according to the binding state and/or number of the nanoparticle and a reaction principle of the double-antibody sandwich structure. As shown in FIG. 4, in an embodiment of the present application, a threshold of the intensity of the electrical pulse signal can be analyzed to determine a charge state of the complex particle, and a minute fluctuation of the electrical pulse signal peak can be analyzed to determine whether the nanoparticle is bound on the complex particle, as well as the binding number. For example, in FIG. 4, when two nanoparticles are bound on the complex particle, the electrical pulse signal is reflected by two peaks; when no nanoparticle is bound on the complex particle, the electrical pulse signal is reflected by absence of peak; and when three nanoparticles are bound on the complex particle, the electrical pulse signal is reflected by three peaks.

[0058] In another embodiment of the present application, the micro-nano pore device is a device having a micropore-bearing double-layer film structure. As shown in FIG. 5, the device comprises two layers of micropore-bearing nano film (micropore 1 and micropore 2 in the figure) spaced apart by a set distance. The micropores on the two nano films are disposed opposite to each other and communicate the cavities (cavity 1 and cavity 2 in the figure) at non-opposing sides of the nano films. The cavities are filled with an electrolyte and are respectively provided with an electrode for maintaining ion transport. When the protein-magnetic bead complex passes through the two micropores successively in the form of a single particle, a pair of electrical pulse signals having a time interval are generated.

[0059] In the above embodiment, the size of the micropores in the device having a double-layer film structure can be in the range from 1 nm to 10 μm, and can be specifically determined according to the requirement of allowing the protein-magnetic bead complex to pass through the micropores successively in the form of a single particle. The nano film can be selected from films prepared from any semiconductor film-forming materials such as silicon nitride, silicon dioxide, silicon, etc., or from high-molecular polymers. The two layers of nano film are spaced apart by a set distance, which can be from 1 nm to 1 μm. The device having a double-layer film structure is equipped with two electrodes, which can be a silver/silver chloride electrode or a platinum electrode. An external electric field is applied to maintain a balanced ion transport, which is reflected by a stable open pore current. When the protein-magnetic bead complex passes through the two micropores successively under the action of the driving force, a pair of electrical pulse signals having a time interval are generated.

[0060] The device having a double-layer film structure according to the present application can be obtained by various suitable methods. For example, in an embodiment of the present application, the device having a double-layer film structure is integrally formed by means of micro-nano processing to control the distance of the two layers of film. The specific principle and steps are as shown in FIG. 6. In step one, a substrate material (silicon, quartz, etc.) having been polished on both sides thereof is provided as a support layer, and a nano film (film-forming layer in the figure) of 1 nm to 10 μm is prepared on the upper surface and the lower surface of the substrate material by means of thermal oxidation or vapor deposition. In step two, previously-prepared photolithography templates are used to define the size and shape of the micropores and the position thereof on the films. A photoresist is coated on the upper and lower surfaces, and the templates are used to perform light exposure and development. The upper and lower templates can be precisely aligned to ensure that the two micropores maintain a micron-scale alignment precision in space. Then, with the unexposed photoresist as a sacrificial layer, the films are etched by means of gas etching to form the micropores. In step three, the support layer is etched by means of wet etching in which an etching solution etches the support layer from the micropores so that the two micropores are in communication with each other.

[0061] As shown in FIG. 7, when complex 1, an unbound magnetic bead (i.e., an immunomagnetic bead not bound with the nanoparticle), and complex 2 pass through the two micropores successively in the form of a single particle, a pair of electrical pulse signals for each of them are generated. The electrical pulse signals have a certain time interval, which reflects the time for passing through the micropores and is associated with electromobility, which in turn is associated with charge state. The time interval between the electrical pulse signals for complex 1 and complex 2 exceeds a set threshold, while the time interval between the electrical pulse signals for the unbound magnetic bead does not exceed the set threshold. FIG. 8 shows true pairs of electrical pulse signals generated when respective complexes pass through the two micropores successively in the form of a single particle. It can be seen that a pair of electrical pulse signals is generated when each complex particle passes through the micropores, and the two electrical pulse signals of the pair have a certain time interval.

[0062] In a preferred embodiment of the present application, the concentration of the complex in the sample is adjusted (for example, smaller than 10 picogram/milliliter) such that the electrical pulse signal interval between respective complexes is much larger than the time interval between the pair of electrical pulse signals, and a pulse intensity threshold of the electrical pulse signals is analyzed so as to filter off the signals generated by immunomagnetic beads not bound with the nanoparticle.

[0063] According to a reaction principle of the double-antibody sandwich structure of the present application, the number of protein molecules captured on the immunomagnetic bead determines the number of the secondary antibody-modified nanoparticle bound thereto, and in turn determines the number of electrical charges carried by the complex, which is associated with electromobility. Therefore, as shown in FIG. 9, a linear relationship exists between the number of captured protein molecules and the electromobility (migration speed). A particle surface potential can be obtained by calculating the electromobility, and then the amount of the to-be-detected protein can be calculated.

[0064] In an embodiment of the present application, the electromobility is calculated according to the following formula:

[00004] $μ = \frac{v (x)}{E (x)} = \frac{\partial v (x)}{\partial E (x)}$

[0065] where μ represents the electromobility, ν(x) represents a speed at which a complex particle passes through the two micropores successively, and E(x) represents an electric field distribution.

[0066] For example, suppose that a distance of the two micropores of 1000 nm, a time interval of 1 millisecond during which a single complex particle passes through the two micropores, and a potential difference of 100 millivolts, then the electromobility can be calculated to be 10.sup.−8 m.sup.2V.sup.−1s.sup.−1.

[0067] Moreover, the particle surface potential (corresponds to the electromobility μ in the following relationship:

[00005] $μ = \frac{ϵξ}{η}$

[0068] where η represents a viscosity of the electrolyte.

[0069] and, the formula for the particle surface potential ξ is as follows:

[00006] $ξ = A \frac{\partial (\frac{1}{t})}{\partial V}$

[0070] where t represents a residence time of the particle in the micropores, A represents a correction factor for each micropore, and V represents a potential difference.

[0071] For example, in an embodiment of the present application, suppose that the correction factor A is 6×10.sup.−6 V.sup.2 s, then the particle surface potential ξ can be calculated to be 60 millivolts.

[0072] In another embodiment of the present application, the amount of the to-be-detected protein in a sample system can be calculated according to the volume/mass state of the complex. In particular, as shown in FIG. 10, the volume/mass state of the complex is positively related to an integral area of the electrical pulse signal. The number of the protein molecules captured on the complex is directly proportional to an increase in volume/mass of the complex relative to the immunomagnetic bead. The number of the protein molecules captured on the complex is be calculated according to a standard curve relationship of the integral area of the electrical pulse signal (which characterizes the volume/mass state of the complex) and the number of the protein molecules captured on the complex, and in turn the amount of the to-be-detected protein in the sample system is calculated. In this embodiment, the volume/mass of the complex represents the volume, the mass, or a combination of both of the complex, because according to a reaction principle of the double-antibody sandwich structure of the present application, the number of protein molecules captured on the immunomagnetic bead determines the number of the secondary antibody-modified nanoparticle bound thereto, and latter in turn determines an increase in the volume or mass of the complex.

[0073] The present application has been described above with reference to specific embodiments, which are merely intended to aid in the understanding of the present application and are not intended to limit the present application thereto. Several simple derivations, variations or substitutions can be made by a person skilled in the art to which the present application pertains in light of the concept of the present application.

METHOD FOR DETECTING MICROPROTEINS IN SAMPLE SYSTEM

Inventors

Cpc classification

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G01N33/54366

PHYSICS

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G01N33/68

PHYSICS

Classification Explorer

G01N2470/04

PHYSICS

Classification Explorer

G01N33/54326

PHYSICS

International classification

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G01N33/543

PHYSICS

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G01N33/68

PHYSICS

Abstract

Claims

Description