WO2024008101A1 - 组织层面单分子分辨率水平目标物质原位成像方法 - Google Patents

组织层面单分子分辨率水平目标物质原位成像方法 Download PDF

Info

Publication number
WO2024008101A1
WO2024008101A1 PCT/CN2023/105772 CN2023105772W WO2024008101A1 WO 2024008101 A1 WO2024008101 A1 WO 2024008101A1 CN 2023105772 W CN2023105772 W CN 2023105772W WO 2024008101 A1 WO2024008101 A1 WO 2024008101A1
Authority
WO
WIPO (PCT)
Prior art keywords
target substance
tissue
deformation
target
level
Prior art date
Application number
PCT/CN2023/105772
Other languages
English (en)
French (fr)
Inventor
辛学刚
黄义虹
Original Assignee
华南理工大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 华南理工大学 filed Critical 华南理工大学
Publication of WO2024008101A1 publication Critical patent/WO2024008101A1/zh

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B11/00—Measuring arrangements characterised by the use of optical techniques
    • G01B11/02—Measuring arrangements characterised by the use of optical techniques for measuring length, width or thickness
    • G01B11/026—Measuring arrangements characterised by the use of optical techniques for measuring length, width or thickness by measuring distance between sensor and object
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B11/00—Measuring arrangements characterised by the use of optical techniques
    • G01B11/16—Measuring arrangements characterised by the use of optical techniques for measuring the deformation in a solid, e.g. optical strain gauge

Definitions

  • the invention relates to the field of biological imaging technology, and specifically relates to a method for in-situ imaging of target substances at a single molecule resolution level at the tissue level.
  • Biomarkers are often used as objects to evaluate specific characteristics of biological physiological processes, pathological processes, or responses to drug interventions.
  • the detection of biomarkers is of great significance in medical diagnosis, clinical research, and new drug development, especially in the diagnosis and treatment of tumors, viral infectious diseases, or other diseases.
  • specific tumor markers will be produced, which can directly reflect the source of the tumor, benign and malignant information, etc.
  • the detection of tumor markers at the tissue level is of great significance to cancer diagnosis and treatment.
  • protein markers play a very important role. Proteins are the executors of biological functions and are the most widely studied and applied biomarkers.
  • the detection method of specific protein markers at the tissue level is usually based on the principle of specific binding of antigens and antibodies to perform immunological detection of protein markers, often using radioactive isotopes, enzymes, colloidal gold and organic fluorescent dyes. Molecules, etc. label the antibodies, use the labeled antibodies to identify the corresponding protein markers on tissue cells, and achieve the purpose of detecting the target protein by qualitatively and quantitatively detecting the labeled antibodies. These methods in the prior art require imaging and judgment through a light microscope.
  • the fluorescence luminescence method is to obtain the overall luminescence of thousands of antigen and antibody proteins through a light microscope. In the case of staining, thousands of them are also obtained through a light microscope. Imaging of the overall luminescence of tens of thousands of stained target proteins. Since light microscopy detects the light intensity of a single protein molecule, existing detection methods based on light microscopy technology cannot obtain information on the target at the scale of a single protein.
  • tissue proteins are usually broken into suspensions for detection.
  • the detection object at that time cannot restore the information at the original tissue level after being broken. Therefore, the methods in the existing technology cannot achieve in-situ imaging information of the target substance at the single molecule resolution level at the tissue level.
  • the purpose of the present invention is to provide a method for in-situ imaging of target substances at the single molecule resolution level at the tissue level, so as to achieve imaging of target substances at the tissue level at the single molecule resolution level.
  • a specific characteristic inducing unit is used to cause a specific interaction between the target substance at the single molecule resolution level in the tissue level and the reference substance contained in the specific characteristic inducing unit;
  • mapping relationship between the target object position and the representation characteristics is generated, and the in-situ imaging result of the target substance is obtained.
  • the specific signature inducing unit is close to and pressed against the tissue level, and the target substance in the tissue level is consistent with the reference contained in the specific signature inducing unit.
  • the substance has a specific effect.
  • the target substance and the reference substance gradually separate from the combination and then change to the complete separation of the target substance and the reference substance.
  • the acquisition unit acquires at least one of the characteristic information on force, electricity or light changes generated during the specific action of the target substance as Characteristic information.
  • the acquisition unit obtains the characterization characteristic information based on the before and after change information of the target substance during the specific action, or the target substance during the specific action.
  • the information generated in the process is compared with the reference sample information to obtain the characteristic characteristic information.
  • the reference sample information is the information obtained when the specific characteristic induction unit acts on the tissue level and does not contain the target substance.
  • the result generation unit obtains tissue level points based on the mapping relationship between the target object position and the characterization characteristics of Equation (1).
  • the signal expression of the imaging position is the characteristic information about force, electricity or light changes generated during the specific action of the target substance acquired by the acquisition unit.
  • Information about the changing characteristics of light for ,in, is the absorption coefficient, is the penetration depth.
  • an antigen-antibody specific interaction occurs between the target substance and the reference substance.
  • the specific signature inducing unit is close to and pressed against the tissue level, and the target substance in the tissue level is consistent with the reference contained in the specific signature inducing unit.
  • the specific binding of substances forms the binding force between the target substance and the reference substance.
  • the binding force between the target substance and the reference substance gradually decreases and then changes to the target substance and the reference substance. Matter is completely separated.
  • the acquisition unit acquires the deformation or binding force between the target substance and the reference substance, and obtains the characteristic information based on the change in deformation or binding force; for ,in, is the elastic coefficient, is the offset;; or
  • the acquisition unit acquires changes in dielectric parameters produced during the interaction between the target substance and the reference substance, and obtains characteristic information based on the changes in dielectric parameters; for ,in, is the electric potential of the electromagnetic field, is the magnetic vector potential, E is the electric field strength, t is time; or
  • the acquisition unit acquires changes in light produced during the interaction between the target substance and the reference substance, and obtains characteristic information based on the changes in light; for ,in, is the absorption coefficient, is the penetration depth.
  • the specific characteristic inducing unit is a deformation biosensor, and the deformation biosensor is provided with a clamping part for being clamped by an external driving device for movement and has a
  • the coupling part is used for the reference substance that specifically interacts with the target protein; the coupling part is an elastic tip-shaped body, which is used to couple a single target substance; the coupling part and the clamping part are provided with smooth surfaces that reflect laser light in any direction. Surface layer.
  • the acquisition unit is provided with a laser emitter, a photosensitive sensor and a characteristic information processor
  • the laser emitter irradiates laser light on the smooth surface layer of the deformation biosensor, and the smooth surface layer reflects the laser light back to the photosensitive sensor
  • the photosensitive sensor detects the offset of the reflected laser on the photosensitive sensor
  • the characteristic information processor is based on the optical path change phenomenon or deformation offset of the deformation sensor at each position obtained during the deformation biosensor acting on the tissue level.
  • at least one kind of information in the force information received by the coupling part of the deformation sensor is used to determine whether there is characteristic information, and to obtain the characteristic information about the change of light or force generated during the specific action of the target substance. and location distribution information ;or
  • the acquisition unit is provided with a dielectric characteristic detection mechanism and a characteristic information processor.
  • the characteristic information processor determines whether there is characteristic information based on the change information of the dielectric parameters at each position obtained during the deformation biosensor acting on the tissue level, and obtains Characteristic information on changes in dielectric properties generated during the specific interaction of the target substance and location distribution information ;or
  • the acquisition unit is provided with a photon feature detection mechanism and a feature information processor.
  • the feature information processor determines whether there is feature information based on the change information of the intensity or frequency of light at each position obtained during the deformation biosensor acting on the tissue level. , obtain characteristic information about the changes in light produced during the specific action of the target substance. and location distribution information .
  • the in-situ imaging method of the target substance at the single molecule resolution level at the tissue level of the present invention uses a specific signature inducing unit to combine the target substance at the single molecule resolution level at the tissue level with the reference substance contained in the specific signature inducing unit.
  • a specific effect occurs; through an acquisition unit, the characteristic information generated by the target substance during the specific effect is obtained; through a result generation unit, a mapping relationship between the position of the target object and the characteristic characteristics is generated, and in-situ imaging of the target substance is obtained result.
  • the imaging method of the present invention can perform target substance imaging at a single molecule resolution level at the tissue level, and solves the technical problem in the prior art that cannot achieve target substance imaging at a tissue level at a single molecule resolution level. This method directly detects tissues, avoiding the shortcomings of the existing technology that require complex preparation of test samples before inspection. It has the characteristics of simple requirements for test samples, fast detection speed, and high detection accuracy.
  • Figure 2 is a schematic structural diagram of a deformation biosensor in the method of the present invention.
  • Figure 3 is a schematic diagram of a deformation detection system in the method of the present invention.
  • Figure 4 is a schematic diagram of another deformation detection system in the method of the present invention.
  • Figure 5 is a flow chart of the method of the present invention.
  • Figure 6 is a schematic diagram of a change detected by the method of the present invention.
  • Figures 1 to 5 include:
  • Deformation biosensor 100 coupling part 110, clamping part 120, reflection point 130, fixed point 140,
  • Driving device 200 laser transmitter 300, photosensitive sensor 400,
  • This embodiment is a method for in-situ imaging of target substances at a single molecule resolution level at the tissue level.
  • the tissue level means that the method of the present invention directly tests biological tissues, and does not require the prior art to process the test sample before detection.
  • the biological tissue tested may be a tissue section or other biological sample to be tested.
  • the general molecular diameter is in the 10 -10 m range, which is 0.1 nanometers. Various substances have different molecular diameters. The diameter of the smaller hydrogen molecule is 0.23 nanometers.
  • the core purpose of the present invention is to be able to detect imaging of a single protein target substance. To detect a single protein target substance, imaging needs to be possible at a resolution scale of 0.1-10 nm. Therefore, the single-molecule resolution level in the present invention refers to the resolution scale of 0.1-10 nm.
  • the target substance can be a single protein or molecule or other substance.
  • Figure 1 is one of the in-situ visualization point maps of tissue proteins obtained by the method of the present invention. From the figure, we can see the distribution and specific location of each target protein (i.e., target substance) represented by triangles in the tissue. From this figure, you can see where the target protein is present in the tissue, that is, where the target protein is in the tissue.
  • target protein i.e., target substance
  • a method for in-situ imaging of target substances at a single molecule resolution level at the tissue level is performed in the following manner:
  • the target substance at the single molecule resolution level in the tissue level interacts specifically with the reference substance contained in the specific characteristic inducing unit;
  • mapping relationship between the target object position and the representation characteristics is generated, and the in-situ imaging result of the target substance is obtained.
  • the specific characteristic inducing unit is set up to induce an interaction with the target substance in the tissue to be detected, so as to create characteristics that are different from those in the tissue that does not contain the target substance because the tissue contains the target substance. Therefore, the specific characteristic inducing unit is provided with a reference substance.
  • the specific characteristic inducing unit is used to detect tissue, when the tissue layer contains the target substance to be measured, the target substance to be measured will have a specific interaction with the reference substance.
  • the so-called specific effect refers to the specific effect between the reference substance and the target substance. This effect does not occur between the reference substance and non-target substances.
  • the specific characteristic inducing unit is close to and pressed against the tissue level, and the target substance in the tissue level interacts specifically with the reference substance contained in the specific characteristic inducing unit.
  • the target substance and the reference substance gradually separate from the combination and then change to the complete separation of the target substance and the reference substance.
  • the specific characteristic inducing unit approaches and presses against the tissue level and then gradually moves away from the tissue level, the characteristics formed by the force between the target substance and the reference substance can be monitored.
  • the change information in this process can be monitored.
  • the acquisition unit acquires at least one of the characteristic information on force, electricity or light changes generated during the specific action of the target substance as the characteristic characteristic information.
  • the acquisition unit can acquire the deformation or force change produced during the interaction between the target substance and the reference material, and obtain characteristic information based on the deformation or force change;
  • the acquisition unit acquires changes in dielectric parameters produced during the interaction between the target substance and the reference substance, and obtains characteristic information based on the changes in dielectric parameters;
  • the acquisition unit acquires changes in light produced during the interaction between the target substance and the reference substance, and obtains characteristic information based on the changes in light.
  • the acquisition unit obtains characteristic information based on the before and after change information generated by the target substance during the specific effect, or compares the information generated by the target substance during the specific effect with the reference sample information to obtain the characteristic information.
  • the sample information is the information obtained when the specific characteristic inducing unit acts on the tissue level that does not contain the target substance.
  • the result generation unit obtains the organizational level points based on the mapping relationship between the target object position and the representation features of Equation (1).
  • the result generation unit After obtaining the order, after obtaining the characteristic information of the occurrence of the target substance, it means that at this detection position, there is a target position to be detected in the tissue.
  • the result generation unit combines the spatial position of the tissue with this result to obtain the original position of the target substance in the tissue.
  • the location is the in-situ information. Obtain the mapping relationship between a specific target substance and its original location. After all locations in the tissue have been detected, the in-situ imaging image of the target substance in the tissue can be obtained as shown in Figure 1.
  • the imaging method of this embodiment is capable of imaging target substances at a single molecule resolution level at the tissue level, which solves the technical problem in the existing technology of being unable to image target substances at a tissue level at a single molecule resolution level.
  • This method directly detects tissues, avoiding the shortcomings of the existing technology that require complex preparation of test samples before inspection. It has the characteristics of simple requirements for test samples, fast detection speed, and high detection accuracy.
  • a method for in-situ imaging of a target substance at a single molecule resolution level at the tissue level is the same as in Embodiment 1, and also have the following features: an antigen-antibody specific interaction occurs between the target substance and the reference substance.
  • the specific signature inducing unit is close to and pressed against the tissue level, and the target substance in the tissue level is specific to the reference substance contained in the specific signature inducing unit.
  • the combination forms a binding force between the target substance and the reference substance.
  • the binding force between the target substance and the reference substance gradually decreases and then changes to the complete separation of the target substance and the reference substance.
  • the acquisition unit obtains the deformation or binding force between the target substance and the reference substance, and obtains characteristic information based on changes in the deformation or binding force. According to the characteristic information, whether the target substance exists at this site in the tissue is obtained.
  • the result generation unit obtains the organizational level points based on the mapping relationship between the target object position and the representation features of Equation (1).
  • the signal expression of the imaging position is the characteristic information about force, electricity or light changes generated during the specific action of the target substance acquired by the acquisition unit.
  • the acquisition unit can obtain the deformation or binding force between the target substance and the reference substance, and obtain characteristic information based on changes in deformation or binding force; for ,in, is the elastic coefficient, is the offset; or
  • the acquisition unit acquires changes in dielectric parameters produced during the interaction between the target substance and the reference substance, and obtains characteristic information based on the changes in dielectric parameters; for ,in, is the electric potential of the electromagnetic field, is the magnetic vector potential, E is the electric field strength, and t is time; or
  • the acquisition unit acquires changes in light produced during the interaction between the target substance and the reference substance, and obtains characteristic information based on the changes in light; for ,in, is the absorption coefficient, z is the penetration depth.
  • a target substance site map of the entire biological tissue can be obtained.
  • the imaging method of this embodiment is capable of imaging target substances at a single molecule resolution level at the tissue level, which solves the technical problem in the existing technology of being unable to image target substances at a tissue level at a single molecule resolution level.
  • This method directly detects tissues, avoiding the shortcomings of the existing technology that require complex preparation of test samples before inspection. It has the characteristics of simple requirements for test samples, fast detection speed, and high detection accuracy.
  • a method for in-situ imaging of a target substance at a single molecule resolution level at the tissue level is provided.
  • an antigen-antibody specific interaction occurs between the target substance and the reference substance.
  • the target substance is an antigen protein and the specific characteristic inducing unit is provided with an antibody. It should be noted that in actual detection, the corresponding matching relationship can be set as needed, and is not limited to the corresponding method in this embodiment.
  • the specific characteristic inducing unit is a deformation biosensor 100.
  • the deformation biosensor is provided with a clamping portion 120 for being clamped by an external driving device for movement and a structure for generating specificity with the target protein.
  • the coupling part 110 of the acting reference material is integrally connected to the sheet-shaped clamping part 120 , and the coupling part 110 is provided at a side end of the sheet-shaped clamping part 120 .
  • the coupling part 110 is an elastic tip-shaped body, and the tip-shaped body is used to couple a single target substance, usually a single antibody molecule is coupled at the tip of the tip-shaped body.
  • This coupling generally uses passive and covalent attachment to fix the antibody molecule on the substrate, that is, the tip position of the coupling part 110 of the present invention.
  • the pointed coupling part can couple a single antibody molecule.
  • the clamping part 120 is a rigid body or an elastic body and is used for fixing and moving the coupling part. When the clamping part and the coupling part are separated from the tissue, the coupling part will undergo slight deformation due to the existence of specific binding force. When the clamping part 120 is an elastic body, it will also be accompanied by slight deformation smaller than that of the coupling part.
  • the coupling part 110 and the clamping part 120 have smooth surfaces that reflect laser light from any direction.
  • the smooth surface layer is metal plating.
  • the metal coating can reduce the scattering of laser light and reflect more light back to the photosensitive sensor, effectively improving the sensitivity of the deformation sensor.
  • the surfaces of the coupling part 110 and the clamping part 120 reflect laser light, their deformations can be identified by the laser displacement sensor.
  • This deformation biosensor is used in such a way that the tip of the coupling part 110 couples the antibody molecules and fully contacts the antigen molecules on the tissue to cause specific binding.
  • the elastic coupling part deforms slightly. This slight deformation is recognized by the laser displacement sensor because the smooth surface of the coupling part can reflect laser light.
  • the acquisition unit is provided with a laser emitter 300, a photosensitive sensor 400 and a feature information processor 500.
  • the laser emitter irradiates the laser on the smooth surface layer of the deformation biosensor (specifically, in this embodiment, the laser irradiates on the triangular sheet coupling part 110 smooth surface layer), the smooth surface layer reflects the laser back to the photosensitive sensor; the photosensitive sensor detects the offset of the reflected laser light on the photosensitive sensor, as shown in Figure 3.
  • the point on the surface of the coupling part 110 that is irradiated with laser light is defined as a reflection point 130 .
  • the reflection point 130 is not limited to the surface of the coupling part 110, but may also be on the surface of the clamping part 120, as shown in FIG. 4 .
  • the coupling part 110 is in the shape of a small needle and its surface area is not enough to receive the laser from the laser transmitter, the reflection point is set on the clamping part 120 that has the reflected laser and elastic deformation, and the effect of detecting the deformation amount of the deformation sensor is also achieved. .
  • the driving device 200 is connected and fixed to the clamping part 120 to drive the elastic coupling part 110 away from or close to the tissue to be measured 600 .
  • the point where the driving device 200 and the clamping part 120 are connected and fixed is defined as a fixed point 140 .
  • the characteristic information processor 500 can be a computer that converts the offset on the photosensitive sensor into the deformation offset of the deformation sensor according to formula (1) or calculates the force on the coupling part of the deformation sensor according to formula (2);
  • d is the deformation offset of the deformation biosensor
  • D is the offset of the reflected light on the photosensitive sensor after deformation of the deformation biosensor
  • a is the distance between the reflection point on the deformation biosensor and the fixed point of the deformation biosensor.
  • Distance b is the distance between the reflection point on the deformation biosensor and the laser emitter
  • ⁇ is the incident angle of the laser emitted by the laser emitter;
  • the reflection point is the point where the laser of the infrared laser transmitter is irradiated on the deformation biosensor
  • the fixed point is the point where the driving device is connected to the fixed part
  • F is the force exerted by the coupling part of the deformation sensor
  • k is the elastic coefficient of the coupling part of the deformation sensor
  • the feature information processor determines whether there is feature information based on the deformation offset of the deformation sensor at each position obtained during the deformation biosensor acting on the tissue level and the force information on the coupling part of the deformation sensor.
  • the characteristic information processor 500 sends a command to the driving device 200, and the driving coupling part 100 gradually approaches and fully contacts the tissue, and then gradually moves away from the tissue 600.
  • the characteristic information processor 500 controls the laser emitting device 300 to emit infrared laser light to illuminate the illumination point of the coupling part 130 during the movement of the coupling part 100, and at the same time, the reflection point reflects the laser light back to the infrared light sensor 400.
  • the characteristic information processor 500 collects the deflection amount and calculates the deformation amount of the deformation biosensor 100 in combination with formula (1) or calculates it through formula (2) Obtain the force exerted on the coupling part of the deformation sensor. Based on whether the target protein is detected at different points (or locations) on the tissue level, in situ imaging information of a single target protein can be obtained.
  • an elastic biosensor is used.
  • the deformation amount of the elastic biosensor changes.
  • it is determined whether the antibody molecules are Whether the target protein molecules to be tested on the tissue are bound to each other, and combined with the tissue spatial position information, a visual point map of the tissue in situ protein can be obtained. It can perform imaging of target substances at the single molecule resolution level at the tissue level, which solves the technical problem of being unable to achieve target substance imaging at the tissue level at the single molecule resolution level in the existing technology. It has simple requirements for detection samples, fast detection speed, and Features of high detection accuracy.
  • the in-situ imaging method of the target substance at the single molecule resolution level at the tissue level in this embodiment uses a deformation biosensor as a specific signature inducing unit.
  • a deformation biosensor as a specific signature inducing unit.
  • multiple coupling parts are provided. Arranged in a matrix. Each coupling part can specifically bind to a target substance, and by providing multiple coupling parts, the detection efficiency can be improved.
  • This embodiment provides a deformation biosensor in Application Embodiment 3 as a specific characteristic inducing unit, using the laser emitter 300, the photosensitive sensor 400 and the characteristic information processor 500 as the acquisition unit to perform target substance profiling at the single molecule resolution level at the tissue level.
  • Bit imaging method is carried out through the following steps:
  • the washed deformation biosensor is stored in PBS at low temperature
  • the treatment process can adopt one of the following parameter processes: the number of soaking and washing times in absolute ethanol is three times, each time for 5 minutes. The number of washes with ultrapure water is three times, 5 minutes each time. The antibody in the antibody solution is CD117, and the volume is 100uL. The coupling reaction time is 1.5h. The number of PBS washes was three times, 5 min each time.
  • the processing process provides one of the following processing parameters:
  • the volume size of the tissue is 0.1*0.1*0.1 cm 3 - 2*2*2 cm 3 .
  • the pH of the buffer is 7.0-7.6, and its ingredients mainly include sodium hydroxide, potassium hydroxide, sodium dihydrogen phosphate, anhydrous magnesium sulfate, glucose, dextran, adenosine, and glutathione.
  • the infrared laser emitter emits laser light to the surface of the deformation biosensor, and then the laser light is reflected back to the photosensitive sensor to obtain the offset D of the laser on the photosensitive sensor;
  • the processing unit calculates the deformation offset d of the deformation biosensor in real time through formula (1);
  • the driving device drives the elastic coupling part close to the tissue until it contacts the tissue, and records the deformation offset of the deformation biosensor and the displacement of the elastic coupling part in real time during this process;
  • the driving device drives the elastic coupling part away from the tissue and away from the tissue, and records the deformation offset of the deformation biosensor and the displacement of the elastic coupling part in real time during this process;
  • the characteristic information processor uses the deformation offset of the real-time deformation biosensor obtained in step S4 as the x-axis, and the displacement of the elastic coupling part as the y-axis to draw a dot-line graph. Based on whether an inflection point appears in the dot-line graph, the antibody and antigen are judged. Whether the molecule produces specific binding and records the coordinate information of the specific binding point on the tissue;
  • the coordinate information recorded in all S5 steps is drawn on the tissue image in the form of points to form a visual point map of tissue in situ proteins at the single-molecule resolution level.
  • the present invention is a method for visualizing tissue in situ proteins at a single molecule resolution level. Its implementation is as follows: deformation biosensors containing antibodies are prepared in batches through step S1. When there is tissue that needs to be detected, the tissue is first The object is processed according to step S1, and then the deformation biosensor is combined with the tissue to be measured, and its deformation amount is measured according to steps S2 to S3. Then, the specific binding points on the tissue are obtained through steps S4 to S5, and finally all the points are gathered on the tissue image to form a visual scatter plot.
  • the method of the present invention for visualizing tissue in situ proteins at a single molecule resolution level is implemented in the above manner and has the characteristics of rapidity and visualization.
  • This embodiment provides a deformation biosensor in Application Embodiment 3 as a specific characteristic inducing unit, using the laser emitter 300, the photosensitive sensor 400 and the characteristic information processor 500 as the acquisition unit to perform target substance profiling at the single molecule resolution level at the tissue level.
  • Bit imaging method is carried out through the following steps:
  • the washed deformation biosensor is stored in PBS at low temperature
  • the treatment process can adopt one of the following parameter processes: the number of soaking and washing times in absolute ethanol is five times, 8 minutes each time. The number of washes with ultrapure water is three times, 10 minutes each time. The antibody in the antibody solution is CD117, and the volume is 200uL. The coupling reaction time is 2.0h. The number of PBS washes was three times, 5 min each time.
  • the processing process provides one of the following processing parameters:
  • the volume size of the tissue is 0.1*0.1*0.1 cm 3 - 2*2*2 cm 3 .
  • the pH of the buffer is 7.0-7.5, and its ingredients mainly include sodium hydroxide, potassium hydroxide, sodium dihydrogen phosphate, anhydrous magnesium sulfate, glucose, dextran, adenosine, and glutathione.
  • the infrared laser emitter emits laser light to the surface of the deformation biosensor, and then the laser light is reflected back to the photosensitive sensor to obtain the offset D of the laser on the photosensitive sensor;
  • the processing unit calculates the deformation offset d of the deformation biosensor in real time through formula (1);
  • the driving device drives the elastic coupling part close to the tissue until it contacts the tissue, and records the deformation offset of the deformation biosensor and the displacement of the elastic coupling part in real time during this process;
  • the driving device drives the elastic coupling part to move away from and away from the tissue, records the deformation offset of the deformation biosensor and the displacement of the elastic coupling part in real time, and calculates the relationship between the sensor and the biological tissue during the real-time process according to formula (2).
  • the feature information processor uses the distance between the biosensor and the biological tissue obtained in step S4 as the x-axis, and the force F between the sensor and the biological tissue as the y-axis to draw a dot-line diagram, as shown in Figure 6. According to the dot-line diagram When the inflection point appears, it is judged that the antibody has specifically combined with the antigen molecule and the coordinate information of the specific binding point on the tissue is recorded;
  • the coordinate information recorded in all S5 steps is drawn on the tissue image in the form of points to form a visual point map of tissue in situ proteins at the single-molecule resolution level.
  • the present invention is a method for visualizing tissue in situ proteins at a single molecule resolution level. Its implementation is as follows: deformation biosensors containing antibodies are prepared in batches through step S1. When there is tissue that needs to be detected, the tissue is first The object is processed according to step S1, and then the deformation biosensor is combined with the tissue to be measured, and its deformation amount and corresponding stress are measured according to steps S2 to S3. Then, the specific binding points on the tissue are obtained through steps S4 to S5, and finally all the points are gathered on the tissue image to form a visual scatter plot.
  • the method of the present invention for visualizing tissue in situ proteins at a single molecule resolution level is implemented in the above manner and has the characteristics of rapidity and visualization.
  • This embodiment provides a method for in-situ imaging of target substances at the single molecule resolution level at the tissue level.
  • the radio frequency transmitting array emits radio frequency electromagnetic waves in the near field to obtain self-emission-self-reception feedback parameters.
  • the target substance that is, the unit tissue and the reference substance are obtained through the acquisition unit. That is, the changes in reflection parameters produced during the action of radio frequency electromagnetic fields obtain the characteristics that reflect the dielectric parameters of the unit tissue.
  • the acquisition unit obtains the change characteristic information of the dielectric parameters generated during the specific interaction of the target substance.
  • location distribution information Get organizational level points The distribution of the change characteristics of the dielectric parameters produced during the specific action of the corresponding target substance .
  • the result generation unit obtains the organizational level points based on the mapping relationship between the target object position and the representation features of Equation (1).
  • the acquisition unit acquires the changes in dielectric parameters produced during the interaction between the target substance and the reference substance, and obtains characteristic information based on the changes in the dielectric parameters; for ,in, is the electric potential of the electromagnetic field, is the magnetic vector potential, E is the electric field strength, and t is time.
  • the imaging method of the present invention can perform imaging of target substances at a single molecule resolution level at the tissue level, and has the characteristics of simple requirements for detection samples, fast detection speed, and high detection accuracy.
  • the unit target is calibrated with a radioactive biological nuclide and the gamma ray photon emitted by the radioactive nuclide in a single shot is detected.
  • Photon changes include light intensity, frequency changes, etc.
  • the acquisition unit obtains the light intensity and radiation frequency of the target substance.
  • the light intensity and frequency information reflect the distribution changes of the reference substance, and characteristic information is obtained based on the changes in light intensity and frequency;
  • the acquisition unit obtains the change characteristic information of light intensity and frequency generated during the specific action of the target substance. and location distribution information .
  • the result generation unit obtains the organizational level points based on the mapping relationship between the target object position and the representation features of Equation (1). The distribution of the changing characteristics of light produced during the specific interaction of the corresponding target substance ; Get organizational level points The distribution of the change characteristics of light generated during the specific interaction of the corresponding target substance .
  • the acquisition unit acquires changes in light produced during the interaction between the target substance and the reference substance, and obtains characteristic information based on the changes in light; for ,in, is the absorption coefficient, z is the penetration depth.
  • the imaging method of the present invention can perform imaging of target substances at a single molecule resolution level at the tissue level, and has the characteristics of simple requirements for detection samples, fast detection speed, and high detection accuracy. Using light intensity, frequency, etc. as characterization objects can reflect the performance characteristics of the material itself, and the results are accurate.

Landscapes

  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Electrochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

一种组织层面单分子分辨率水平目标物质原位成像方法，通过一特异性征诱发单元使组织层面中的单分子分辨率水平的目标物质与特异性征诱发单元中含有的参照物质发生特异性作用；通过一获取单元获取目标物质在发生特异性作用过程中产生的特征信息；通过一结果生成单元，生成目标对象位置与表征特征之间的映射关系，获得目标物质的原位成像结果。该成像方法能够在组织层面进行单分子分辨率水平的目标物质成像，具有对检测样本要求简单，检测速度快、检测精度高的特点。

Description

组织层面单分子分辨率水平目标物质原位成像方法 技术领域
本发明涉及生物成像技术领域，具体涉及一种组织层面单分子分辨率水平目标物质原位成像方法。
 背景技术
生物标志物通常作为评价生物生理过程、病理过程或对药物干预反应的特异性特征的对象。生物标志物的检测在医学诊断、临床研究、新药开发方面具有重要意义，尤其在肿瘤、病毒感染性疾病或其他疾病的诊断和治疗上具有重要价值。以肿瘤研究为例，肿瘤发生发展过程中，会产生特定的肿瘤标志物，能够直接反映肿瘤的来源、良恶性等信息，检测组织层面的肿瘤标志物对癌症诊断和治疗有重要意义。
组织层面生物标志物中，蛋白标志物具有非常重要的地位。蛋白是生物学功能的执行者，是研究和应用最广泛的生物标志物。现有技术中，对组织层面特定的蛋白标志物的检测方法，通常是依据抗原与抗体特异性结合的原理对蛋白标志物进行免疫学检测，经常利用放射性同位素、酶、胶体金和有机荧光染料分子等对抗体进行标记，利用被标记的抗体，识别组织细胞上相应的蛋白标记物，通过定性和定量检测已标记的抗体而达到检测目标蛋白的目的。现有技术中的这些方法，需要通过光显微镜进行成像判断，例如荧光发光法是通过光显微镜获取成千上万个抗原抗体蛋白整体的发光情况成像，染色的情况下也是通过光显微镜获取成千上万个染色的目标蛋白整体的发光情况成像。由于光显微镜检测到单个蛋白分子的光强度，现有技术中基于光学显微镜技术的检测方法，无法得到单个蛋白尺度的目标物的情况信息。
此外，现有技术中的检测方法，因为要在抗原抗体蛋白结合的复合物分子达到一定的浓度（含量）才能检测到光强度信息，通常将组织蛋白破碎做成悬液的形式进行检测，此时的检测对象经过破碎无法还原原始组织层面的信息情况，故，现有技术中的方法，无法实现组织层面单分子分辨率水平目标物质的原位成像信息。
组织层面生物物质单分子分辨率水平的检测，一直是没有解决的重要技术问题。因此，提供一种组织层面单分子分辨率水平目标物质原位成像方法以克服此技术难题甚为必要。
 发明内容
本发明的目的在于提供一种组织层面单分子分辨率水平目标物质原位成像方法，实现在单分子分辨率水平下的组织层面目标物质成像。
本发明的目的通过下述技术方案实现：
提供一种组织层面单分子分辨率水平目标物质原位成像方法，通过如下方式进行：
通过一特异性征诱发单元使组织层面中的单分子分辨率水平的目标物质与所述特异性征诱发单元中含有的参照物质发生特异性作用；
通过一获取单元获取目标物质在发生特异性作用过程中产生的特征信息； 
通过一结果生成单元，生成目标对象位置与表征特征之间的映射关系，获得目标物质的原位成像结果。
优选的，上述的组织层面单分子分辨率水平目标物质原位成像方法，特异性征诱发单元接近并贴压于组织层面，组织层面中的目标物质与所述特异性征诱发单元中含有的参照物质发生特异性作用，在特异性征诱发单元远离组织层面的过程中，目标物质与参照物质之间从结合逐渐分离再变化到目标物质与参照物质完全分离。
优选的，上述的组织层面单分子分辨率水平目标物质原位成像方法，获取单元获取目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征信息中的至少一种作为表征特征信息。
优选的，上述的组织层面单分子分辨率水平目标物质原位成像方法，获取单元根据目标物质在发生特异性作用过程中产生的前后变化信息获得表征特征信息，或者将目标物质在发生特异性作用过程中产生的信息与参照样本信息进行比对得到表征特征信息，所述参照样本信息是特异性征诱发单元作用于组织层面的不含有目标物质时得到的信息。
优选的，上述的组织层面单分子分辨率水平目标物质原位成像方法，所述结果生成单元根据式（1）的目标对象位置与表征特征之间的映射关系，得到组织层面点 对应的目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征的分布 ；
……式（1）；
其中， 表示原位成像像素的位置信息，本处用笛卡尔坐标系表达， 是横坐标,  是纵坐标。
是对应的 成像位置的信号表达，即获取单元获取的目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征信息。
具体的，关于力的变化特征信息， 为 ，其中， 为弹性系数， 为偏移量；
关于电的变化特征信息： 为 ，其中， 为电磁场的电势， 为磁向量势， E为电场强度，t为时间；
关于光的变化特征信息： 为 ，其中， 为吸收系数， 为穿透深度。优选的，上述的组织层面单分子分辨率水平目标物质原位成像方法，目标物质与参照物质之间发生抗原-抗体特异性作用。
优选的，上述的组织层面单分子分辨率水平目标物质原位成像方法，特异性征诱发单元接近并贴压于组织层面，组织层面中的目标物质与所述特异性征诱发单元中含有的参照物质发生特异性结合形成目标物质与参照物质之间的结合力，在特异性征诱发单元远离组织层面的过程中，目标物质与参照物质之间从结合力逐渐减小再变化到目标物质与参照物质完全分离。
优选的， 获取单元获取目标物质与参照物质之间的形变或者结合力，根据形变或者结合力的变化获得特征信息； 为 ，其中， 为弹性系数， 为偏移量；； 或者
获取单元获取目标物质与参照物质作用过程中产生的介电参数变化，根据介电参数的变化获得特征信息； 为 ，其中， 为电磁场的电势， 为磁向量势， E为电场强度，t为时间；；或者
获取单元获取目标物质与参照物质作用过程中产生的光的变化，根据光的变化获得特征信息； 为 ，其中， 为吸收系数， 为穿透深度。
优选的，上述的组织层面单分子分辨率水平目标物质原位成像方法，特异性征诱发单元为形变生物传感器，形变生物传感器设置有用于被外部驱动装置夹持以进行移动的夹持部和具有用于与目标蛋白发生特异性作用的参照物质的耦合部；耦合部为具有弹性的尖状体，尖状体用于耦合单个目标物质；耦合部和夹持部设置有反射任何方向激光的光滑面层。
优选的，获取单元设置有激光发射器、光敏感应器和特征信息处理器，激光发射器将激光照射在形变生物传感器的光滑面层，所述光滑面层将激光反射回光敏感应器；所述光敏感应器检测反射回来的激光在光敏感应器上的偏移量；所述特征信息处理器根据形变生物传感器作用于组织层面过程中获得的各个位置处形变传感器的光路变化现象或者形变偏移量或者形变传感器耦合部受到的作用力信息中的至少一种信息判断是否存在特征信息，获得目标物质发生特异性作用过程中产生的关于光的或者力的变化特征信息 和位置分布信息 ；或者
获取单元设置有介电特征检测机构和特征信息处理器，所述特征信息处理器根据形变生物传感器作用于组织层面过程中获得的各个位置处的介电参数的变化信息判断是否存在特征信息，获得目标物质发生特异性作用过程中产生的关于介电性能的变化特征信息 和位置分布信息 ；或者
获取单元设置有光子特征检测机构和特征信息处理器，所述特征信息处理器根据形变生物传感器作用于组织层面过程中获得的各个位置处的光的光强或频率的变化信息判断是否存在特征信息，获得目标物质发生特异性作用过程中产生的关于光的变化特征信息 和位置分布信息 。
本发明的组织层面单分子分辨率水平目标物质原位成像方法，通过一特异性征诱发单元使组织层面中的单分子分辨率水平的目标物质与所述特异性征诱发单元中含有的参照物质发生特异性作用；通过一获取单元获取目标物质在发生特异性作用过程中产生的特征信息；通过一结果生成单元，生成目标对象位置与表征特征之间的映射关系，获得目标物质的原位成像结果。本发明的成像方法能够在组织层面进行单分子分辨率水平的目标物质成像，解决了现有技术中无法实现单分子分辨率水平下的组织层面目标物质成像的技术难题。该方法直接对组织进行检测，避免了现有技术中需要对检测样本进行复杂制备后才能进行检查的缺陷，具有对检测样本要求简单，检测速度快、检测精度高的特点。
 附图说明
图1是本发明方法获得的一组织蛋白原位可视化点位图，比例尺=1μm。
图2是本发明方法中的一种形变生物传感器的结构示意图。
图3是本发明方法中的一种形变检测系统的示意图。
图4是本发明方法中的另一形变检测系统的示意图。
图5是本发明的方法的流程图。
图6是本发明的方法的检测到的一种变化示意图。
在图1至图5中，包括：
形变生物传感器100、耦合部110、夹持部120、反射点130、固定点140、
驱动装置200、激光发射器300、光敏传感器400、
处理单元500、组织物600。
 具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1。
本实施例是一种组织层面单分子分辨率水平目标物质原位成像方法，组织层面是指本发明的方法直接对生物组织进行测试，不需要现有技术中对测试样品进行处理后才能进行检测，测试的生物组织可以是组织切片或者其它待测试的生物样本。
一般的分子直径大小是10 -10m级别，即0.1纳米。各种不同的物质，分子直径不同，较小的氢分子直径是0.23纳米。本发明的核心目的是为了能够检测到单个蛋白目标物质的成像，要检测到单个蛋白目标物质，需要在0.1-10nm的分辨率尺度能够成像。因此，本发明中的单分子分辨率水平指0.1-10nm的分辨率尺度。目标物质可以是单个蛋白或者分子或者其它物质。
图1是通过本发明的方法获得的其中一幅组织蛋白原位可视化点位图，从图中可以看到三角形代表的各个目标蛋白（即目标物质）在组织中的分布及具体的位置。通过此图可以看到，在组织中哪个位置存在目标蛋白，即在目标蛋白在组织中的位置。
本实施例的一种组织层面单分子分辨率水平目标物质原位成像方法，通过如下方式进行：
通过一特异性征诱发单元使组织层面中的单分子分辨率水平的目标物质与特异性征诱发单元中含有的参照物质发生特异性作用；
通过一获取单元获取目标物质在发生特异性作用过程中产生的特征信息； 
通过一结果生成单元，生成目标对象位置与表征特征之间的映射关系，获得目标物质的原位成像结果。
特异性征诱发单元的设置，是为了诱导与待检测的组织中的目标物质发生作用，以创造能够检测到因为组织中因为含有目标物质而产生有别于组织中不含目标物质的特性。因此，特异性征诱发单元设置有参照物质，利用特异性征诱发单元检测组织时，当组织层面中含有目标待测物质时，待测的目标物质将与参照物质发生特异性作用。所谓特异性作用是指参照物质与目标物质之间特异性发生的作用，参照物质与非目标物质之间不发生这种作用。
具体的，特异性征诱发单元接近并贴压于组织层面，组织层面中的目标物质与特异性征诱发单元中含有的参照物质发生特异性作用，在特异性征诱发单元远离组织层面的过程中，目标物质与参照物质之间从结合逐渐分离再变化到目标物质与参照物质完全分离。
特异性征诱发单元接近并贴压于组织层面再到逐渐远离组织层面的过程中，目标物质与参照物质之间产生作用力所形成的特征，可以对此过程中的变化信息进行监测。获取单元获取目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征信息中的至少一种作为表征特征信息。
例如，获取单元可获取目标物质与参照物质作用过程中产生的形变或者受力变化，根据形变或者受力的变化获得特征信息；或者
获取单元获取目标物质与参照物质作用过程中产生的介电参数变化，根据介电参数的变化获得特征信息；或者
获取单元获取目标物质与参照物质作用过程中产生的光的变化，根据光的变化获得特征信息。
具体的，获取单元根据目标物质在发生特异性作用过程中产生的前后变化信息获得特征信息，或者将目标物质在发生特异性作用过程中产生的信息与参照样本信息进行比对得到特征信息，参照样本信息是特异性征诱发单元作用于组织层面的不含有目标物质时得到的信息。
结果生成单元根据式（1）的目标对象位置与表征特征之间的映射关系，得到组织层面点 对应的目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征的分布 ；
……式（1）；
其中， 表示原位成像像素的位置信息，本处用笛卡尔坐标系表达， 是横坐标， 是纵坐标。
获取单后获取到目标物质发生的特征信息后，说明在此检测位置处，组织中存在待检测的目标位置，结果生成单元根据此结果结合组织的空间位置，得到该目标物质在组织中的原始位置即原位信息。得到某个特定的目标物质与其原始位置的映射关系。当对组织中的所有位置检测完毕后，就可以获得如图1所示的组织中的目标物质的原位成像图。
本实施例的成像方法能够在组织层面进行单分子分辨率水平的目标物质成像，解决了现有技术中无法实现单分子分辨率水平下的组织层面目标物质成像的技术难题。该方法直接对组织进行检测，避免了现有技术中需要对检测样本进行复杂制备后才能进行检查的缺陷，具有对检测样本要求简单，检测速度快、检测精度高的特点。
实施例2。
一种组织层面单分子分辨率水平目标物质原位成像方法，其它特征与实施例1相同，还具有如下特征：目标物质与参照物质之间发生抗原-抗体特异性作用。
在该组织层面单分子分辨率水平目标物质原位成像方法中，特异性征诱发单元接近并贴压于组织层面，组织层面中的目标物质与特异性征诱发单元中含有的参照物质发生特异性结合形成目标物质与参照物质之间的结合力，在特异性征诱发单元远离组织层面的过程中，目标物质与参照物质之间从结合力逐渐减小再变化到目标物质与参照物质完全分离。
获取单元获取目标物质与参照物质之间的形变或者结合力，根据形变或者结合力的变化获得特征信息。根据特征信息得到组织中此位点是否存在目标物质。
结果生成单元根据式（1）的目标对象位置与表征特征之间的映射关系，得到组织层面点 对应的目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征的分布 ；
……式（1）；
其中， 、 分别是获取单元获取的目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征信息和位置分布信息， 是位置坐标。
是对应的 成像位置的信号表达，即获取单元获取的目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征信息。
获取单元可获取目标物质与参照物质之间的形变或者结合力，根据形变或者结合力的变化获得特征信息； 为 ，其中， 为弹性系数， 为偏移量； 或者
获取单元获取目标物质与参照物质作用过程中产生的介电参数变化，根据介电参数的变化获得特征信息； 为 ，其中， 为电磁场的电势， 为磁向量势， E为电场强度，t为时间；或者
获取单元获取目标物质与参照物质作用过程中产生的光的变化，根据光的变化获得特征信息； 为 ，其中， 为吸收系数， z为穿透深度。
依次采用相同的方式对组织中不同位点进行检测，根据每个位点的位置信息及是否存在目标物质结果，可以得到整个生物组织的目标物质位点图。
本实施例的成像方法能够在组织层面进行单分子分辨率水平的目标物质成像，解决了现有技术中无法实现单分子分辨率水平下的组织层面目标物质成像的技术难题。该方法直接对组织进行检测，避免了现有技术中需要对检测样本进行复杂制备后才能进行检查的缺陷，具有对检测样本要求简单，检测速度快、检测精度高的特点。
实施例3。
一种组织层面单分子分辨率水平目标物质原位成像方法，本实施例中目标物质与参照物质之间发生抗原-抗体特异性作用。
本实施例以目标物质为抗原蛋白、特异性征诱发单元设置有抗体为例进行说明。需要说明的是，实际检测中，可以根据需要设置对应的匹配关系，不局限于本实施例的对应方式。
具体的，特异性征诱发单元为形变生物传感器100，如图2所示，形变生物传感器设置有用于被外部驱动装置夹持以进行移动的夹持部120和具有用于与目标蛋白发生特异性作用的参照物质的耦合部110。耦合部110与片状夹持部120一体式连接，耦合部110设置于片状的夹持部120的侧端。耦合部110为具有弹性的尖状体，尖状体用于耦合单个目标物质，通常在尖状体的尖端处耦合单个抗体分子。
该耦合一般采用被动和共价附着的方式将抗体分子固定在底物上，即本发明的耦合部110的尖端位置。尖状的耦合部，可以耦合单个抗体分子。当耦合固定好的抗体分子与组织物上的抗原充分接触时，产生特异性结合力，该结合力在耦合部与组织物分离过程中，使耦合部产生微小形变。
夹持部120为刚性体或者弹性体，用于耦合部的固定以及移动。当夹持部带着耦合部与组织物分离过程中，因为特异性结合作用力的存在，耦合部会发生微小形变。当夹持部120为弹性体时，也会伴随着小于耦合部的微小的形变。
耦合部110和夹持部120具有反射任何方向激光的光滑面层。优选光滑面层为金属镀层。金属镀层可以减少激光的散射，将更多光量反射回光敏传感器，有效提高形变传感器的灵敏度。
当耦合部110和夹持部120表面反射激光时，其形变能被激光位移传感器识别。
该形变生物传感器，其使用是这样的，耦合部110的尖端耦合抗体分子，与组织物上的抗原分子充分接触发生特异性结合，当耦合部与组织物分离过程中，因为结合力的存在使具有弹性的耦合部会发生微小形变。该微小形变因为耦合部光滑表面能反射激光，被激光位移传感器识别到形变。
获取单元设置有激光发射器300、光敏感应器400和特征信息处理器500，激光发射器将激光照射在形变生物传感器的光滑面层（本实施例中具体是将激光照射在三角形片状耦合部110的光滑面层），光滑面层将激光反射回光敏感应器；光敏感应器检测反射回来的激光在光敏感应器上的偏移量，如图3所示。
将激光照射在耦合部110的表面上的点定义为反射点130。需要说明的是, 反射点130不局限于在耦合部110的表面，也可以是在夹持部120表面，如图4所示。当耦合部110形状为细小的针形，其表面积不足以接收激光发射器的激光时，将反射点设置在具有反射激光和弹性形变的夹持部120上，同样达到检测形变传感器形变量的效果。
驱动装置200与夹持部120连接固定，驱动弹性耦合部110远离或者靠近待测组织物600。将驱动装置200与夹持部120连接固定的点定义为固定点140。
特征信息处理器500可以为计算机，根据公式（1）将光敏感应器上的偏移量换算为形变传感器的形变偏移量或者根据公式（2）计算得到形变传感器耦合部受到的作用力；
……（1）；
其中， d是形变生物传感器的形变偏移量， D是形变生物传感器发生形变后反射光在光敏传感器上的偏移量，a是形变生物传感器上的反射点与形变生物传感器固定点之间的距离，b是形变生物传感器上反射点与激光发射器之间的距离， θ是激光发射器发射激光的入射角；
其中，反射点是红外激光发射器的激光照射在形变生物传感器上的点，固定点是驱动装置与固定部连接的点；
F=kd    ……（2）；
其中， F为形变传感器耦合部受到的作用力， k为形变传感器耦合部的弹性系数。
特征信息处理器根据形变生物传感器作用于组织层面过程中获得的各个位置处形变传感器的形变偏移量及形变传感器耦合部受到的作用力信息判断是否存在特征信息。
本发明的方法，特征信息处理器500向驱动装置200发送命令，驱动耦合部100逐渐靠近并充分接触组织物，再逐渐离开组织物600。特征信息处理器500控制激光发射装置300在耦合部100移动过程中发射红外激光照射在耦合部130的照射点上，同时该反射点将激光反射回红外光敏感应器400。因为耦合部100的形变导致激光在红外光敏感应器400上发生偏移，特征信息处理器500收集该偏移量并结合公式（1）计算形变生物传感器100的形变量或者通过公式（2）计算得到形变传感器耦合部受到的作用力。根据组织层面不同点位（或者称位置）处是否检测到目标蛋白，从而获得单个目标蛋白的原位成像信息。
本实施例中使用弹性生物传感器，当弹性生物传感器上的抗体分子与组织上待测目的蛋白分子发生结合时，弹性生物传感器的形变量发生变化，通过检测弹性生物传感器的形变，判断抗体分子与组织上待测目的蛋白分子是否发生结合，结合组织空间位置信息，获得组织原位蛋白可视化点位图。能够在组织层面进行单分子分辨率水平的目标物质成像，解决了现有技术中无法实现单分子分辨率水平下的组织层面目标物质成像的技术难题，具有对检测样本要求简单，检测速度快、检测精度高的特点。
实施例4。
本实施例的组织层面单分子分辨率水平目标物质原位成像方法，使用形变生物传感器作为特异性征诱发单元，为了提高检测效率，本实施例中，耦合部设置有多个，多个耦合部呈矩阵式排列。每个耦合部可以与一个目标物质特异性结合，通过设置多个耦合部，可以提高检测效率。
实施例5。
本实施例提供一种应用实施例3形变生物传感器作为特异性征诱发单元、以激光发射器300、光敏感应器400和特征信息处理器500作为获取单元进行组织层面单分子分辨率水平目标物质原位成像方法，通过以下步骤进行：
S1：耦合部耦合抗体分子
将形变生物传感器用无水乙醇浸泡洗涤，再用超纯水洗涤；
将超纯水洗涤后的形变生物传感器置于抗体溶液中偶联反应，再用PBS洗涤；
洗涤后的形变生物传感器置于PBS中低温保存；
处理过程可采用如下的其中一种参数工艺：无水乙醇浸泡洗涤次数为三次，每次5min。超纯水的洗涤次数为三次，每次5min。抗体溶液中的抗体为 CD117，体积为100uL。偶联反应的时间为1.5h。PBS洗涤次数为三次，每次5min。
S2：组织物的前处理
裁取一定体积的新鲜的组织样本或者冰冻的组织样本，置于缓冲液中静置待用。
处理过程提供其中一种下方式的处理参数： 组织物的体积大小为0.1*0.1*0.1 cm 3- 2*2*2 cm 3。缓冲液的pH为7.0-7.6，成分主要包括氢氧化钠、氢氧化钾、磷酸二氢钠、无水硫酸镁、葡萄糖、葡聚糖、腺苷、谷胱甘肽。
S3：传感器形变量的实时检测
将形变传感器平行正对待检测组织表面并保持距离； 
红外激光发射器向形变生物传感器表面发出激光，然后激光被反射回光敏传感器，得到激光在光敏感应器上的偏移量D；
记录形变生物传感器上的反射点与形变生物传感器固定点之间的距离a，记录形变生物传感器上反射点与激光发射器之间的距离b;
处理单元通过公式（1）实时计算形变生物传感器的形变偏移量d；
S4：形变生物传感器与组织物进行接触和分离
驱动装置驱动弹性耦合部向组织物靠近直至接触，实时记录该过程的形变生物传感器的形变偏移量和弹性耦合部的位移量；
驱动装置驱动弹性耦合部向组织物远离并脱离组织物，实时记录该过程的形变生物传感器的形变偏移量和弹性耦合部的位移量；
S5: 特异性结合点坐标的获取
特征信息处理器以S4步骤获得的实时的形变生物传感器的形变偏移量为x轴，弹性耦合部的位移量为y轴作点线图，根据点线图中是否出现拐点，判断抗体与抗原分子是否产生特异性结合并记录组织物上有特异性结合点的坐标信息；
S6：绘制组织物图像上特异性结合点
调整弹性耦合部与组织物在水平方向的相对位置，重复S3至S5步骤，直至将组织物上以固定距离分布的所有点检测完；
将所有S5步骤记录的坐标信息，以点的形式绘制在组织物图像上，形成单分子分辨率水平的组织原位蛋白可视化点位图。
本发明的一种单分子分辨率水平的组织原位蛋白可视化的方法，其实施方式是这样的，通过S1步骤批量制备含有抗体的形变生物传感器，当有需要检测的组织物时，先对组织物按S1步骤处理，再将形变生物传感器与待测组织物结合，按S2至S3步骤测其形变量。接着通过S4至S5步骤获取组织物上具有特异性结合的点，最后将所有的点汇集在组织物图像上，形成可视化的散点图。
本发明的一种单分子分辨率水平的组织原位蛋白可视化的方法，通过上述方式实施，具有快速、可视化的特点。
实施例6。
本实施例提供一种应用实施例3形变生物传感器作为特异性征诱发单元、以激光发射器300、光敏感应器400和特征信息处理器500作为获取单元进行组织层面单分子分辨率水平目标物质原位成像方法，通过以下步骤进行：
S1：耦合部耦合抗体分子
将形变生物传感器用无水乙醇浸泡洗涤，再用超纯水洗涤；
将超纯水洗涤后的形变生物传感器置于抗体溶液中偶联反应，再用PBS洗涤；
洗涤后的形变生物传感器置于PBS中低温保存；
处理过程可采用如下的其中一种参数工艺：无水乙醇浸泡洗涤次数为五次，每次8min。超纯水的洗涤次数为三次，每次10min。抗体溶液中的抗体为 CD117，体积为200uL。偶联反应的时间为2.0h。PBS洗涤次数为三次，每次5min。
S2：组织物的前处理
裁取一定体积的新鲜的组织样本或者冰冻的组织样本，置于缓冲液中静置待用。
处理过程提供其中一种下方式的处理参数： 组织物的体积大小为0.1*0.1*0.1 cm 3- 2*2*2 cm 3。缓冲液的pH为7.0-7.5，成分主要包括氢氧化钠、氢氧化钾、磷酸二氢钠、无水硫酸镁、葡萄糖、葡聚糖、腺苷、谷胱甘肽。
S3：传感器形变量的实时检测
将形变传感器平行正对待检测组织表面并保持距离； 
红外激光发射器向形变生物传感器表面发出激光，然后激光被反射回光敏传感器，得到激光在光敏感应器上的偏移量D；
记录形变生物传感器上的反射点与形变生物传感器固定点之间的距离a，记录形变生物传感器上反射点与激光发射器之间的距离b;
处理单元通过公式（1）实时计算形变生物传感器的形变偏移量d；
再根据公式（2）实时计算传感器与生物组织之间的作用力F。
S4：形变生物传感器与组织物进行接触和分离
驱动装置驱动弹性耦合部向组织物靠近直至接触，实时记录该过程的形变生物传感器的形变偏移量和弹性耦合部的位移量；
驱动装置驱动弹性耦合部向组织物远离并脱离组织物，实时记录该过程的形变生物传感器的形变偏移量和弹性耦合部的位移量并根据公式（2）计算实时过程中传感器与生物组织之间的作用力F；
S5: 特异性结合点坐标的获取
特征信息处理器以S4步骤获得的生物传感器与生物组织之间的距离为x轴，传感器与生物组织之间的作用力F为y轴作点线图，如图6所示，根据点线图中出现的拐点，判断抗体与抗原分子产生了特异性结合并记录组织物上有特异性结合点的坐标信息；
S6：绘制组织物图像上特异性结合点
调整弹性耦合部与组织物在水平方向的相对位置，重复S3至S5步骤，直至将组织物上以固定距离分布的所有点检测完；
将所有S5步骤记录的坐标信息，以点的形式绘制在组织物图像上，形成单分子分辨率水平的组织原位蛋白可视化点位图。
本发明的一种单分子分辨率水平的组织原位蛋白可视化的方法，其实施方式是这样的，通过S1步骤批量制备含有抗体的形变生物传感器，当有需要检测的组织物时，先对组织物按S1步骤处理，再将形变生物传感器与待测组织物结合，按S2至S3步骤测其形变量及对应力。接着通过S4至S5步骤获取组织物上具有特异性结合的点，最后将所有的点汇集在组织物图像上，形成可视化的散点图。
本发明的一种单分子分辨率水平的组织原位蛋白可视化的方法，通过上述方式实施，具有快速、可视化的特点。
实施例7。
本实施例提供一种组织层面单分子分辨率水平目标物质原位成像方法，射频发射阵列近场发射射频电磁波，获取自发射-自接收反馈参数，通过获取单元获取目标物质即单元组织与参照物质即射频电磁场作用过程中产生的反射参数变化，得到反映单元组织的介电参数的特征。
采用开端同轴探头法，获取单元获取目标物质发生特异性作用过程中产生的介电参数的变化特征信息 和位置分布信息 得到组织层面点 对应的目标物质发生特异性作用过程中产生的介电参数的变化特征的分布 。
具体的，结果生成单元根据式（1）的目标对象位置与表征特征之间的映射关系，得到组织层面点 对应的目标物质发生特异性作用过程中产生的关于电的变化特征的分布
……式（1）。
作为方式之一，获取单元获取目标物质与参照物质作用过程中产生的介电参数变化，根据介电参数的变化获得特征信息； 为 ，其中， 为电磁场的电势， 为磁向量势， E为电场强度，t为时间。
本发明的成像方法能够在组织层面进行单分子分辨率水平的目标物质成像，具有对检测样本要求简单，检测速度快、检测精度高的特点。
实施例8。
将单元目标用放射性生物核素进行标定，检测放射性核素单次辐射的伽马射线光子。光子变化包括光强、频率变化等。获取单元获取目标物质的光强、辐射频率，该光强和频率信息反映参照物质的分布变化，根据光强和频率的变化获得特征信息；
采用光强和频率探测，获取单元获取目标物质发生特异性作用过程中产生的光强、频率的变化特征信息 和位置分布信息 。结果生成单元根据式（1）的目标对象位置与表征特征之间的映射关系，得到组织层面点 对应的目标物质发生特异性作用过程中产生的关于光的变化特征的分布 ；得到组织层面点 对应的目标物质发生特异性作用过程中产生的光的变化特征的分布 。
……式（1）。作为方式之一，获取单元获取目标物质与参照物质作用过程中产生的光的变化，根据光的变化获得特征信息； 为 ，其中， 为吸收系数， z为穿透深度。
本发明的成像方法能够在组织层面进行单分子分辨率水平的目标物质成像，具有对检测样本要求简单，检测速度快、检测精度高的特点。以光强、频率等作为表征对象，能够反映物质本身的性能特性，结果准确。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 一种组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，通过如下方式进行：
    通过一特异性征诱发单元使组织层面中的单分子分辨率水平的目标物质与所述特异性征诱发单元中含有的参照物质发生特异性作用；
    通过一获取单元获取目标物质在发生特异性作用过程中产生的特征信息； 
    通过一结果生成单元，生成目标对象位置与表征特征之间的映射关系，获得目标物质的原位成像结果。
  2. 根据权利要求1所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，特异性征诱发单元接近并贴压于组织层面，组织层面中的目标物质与所述特异性征诱发单元中含有的参照物质发生特异性作用，在特异性征诱发单元远离组织层面的过程中，目标物质与参照物质之间从结合逐渐分离再变化到目标物质与参照物质完全分离。
  3. 根据权利要求2所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，获取单元获取目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征信息中的至少一种作为表征特征信息。
  4. 根据权利要求3所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，获取单元根据目标物质在发生特异性作用过程中产生的前后变化信息获得表征特征信息，或者将目标物质在发生特异性作用过程中产生的信息与参照样本信息进行比对得到表征特征信息，所述参照样本信息是特异性征诱发单元作用于组织层面的不含有目标物质时得到的信息。
  5. 根据权利要求1至4任意一项所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，
    所述结果生成单元根据式（1）的目标对象位置与表征特征之间的映射关系，得到组织层面点 对应的目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征的分布 ；
    ……式（1）；
    其中， 表示原位成像像素的位置信息，本处用笛卡尔坐标系表达， 是横坐标， 是纵坐标；
    是对应的 成像位置的信号表达，即获取单元获取的目标物质发生特异性作用过程中产生的关于力的、电的或者光的变化特征信息。
  6. 根据权利要求1至4任意一项所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，目标物质与参照物质之间发生抗原-抗体特异性作用。
  7. 根据权利要求5所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，特异性征诱发单元接近并贴压于组织层面，组织层面中的目标物质与所述特异性征诱发单元中含有的参照物质发生特异性结合形成目标物质与参照物质之间的结合力，在特异性征诱发单元远离组织层面的过程中，目标物质与参照物质之间从结合力逐渐减小再变化到目标物质与参照物质完全分离。
  8.  根据权利要求7所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，
    获取单元获取目标物质与参照物质之间的形变或者结合力，根据形变或者结合力的变化获得特征信息； 为 ，其中， 为弹性系数， 为偏移量； 或者
    获取单元获取目标物质与参照物质作用过程中产生的介电参数变化，根据介电参数的变化获得特征信息； 为 ，其中， 为电磁场的电势， 为磁向量势，E为电场强度，t为时间；或者
    获取单元获取目标物质与参照物质作用过程中产生的光的变化，根据光的变化获得特征信息； 为 ，其中， 为吸收系数， z为穿透深度。
  9. 根据权利要求8所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，特异性征诱发单元为形变生物传感器，形变生物传感器设置有用于被外部驱动装置夹持以进行移动的夹持部和具有用于与目标蛋白发生特异性作用的参照物质的耦合部；耦合部为具有弹性的尖状体，尖状体用于耦合单个目标物质；耦合部和夹持部设置有反射任何方向激光的光滑面层。
  10.  10. 根据权利要求8所述的组织层面单分子分辨率水平目标物质原位成像方法，其特征在于，
    获取单元设置有激光发射器、光敏感应器和特征信息处理器，激光发射器将激光照射在形变生物传感器的光滑面层，所述光滑面层将激光反射回光敏感应器；所述光敏感应器检测反射回来的激光在光敏感应器上的偏移量；所述特征信息处理器根据形变生物传感器作用于组织层面过程中获得的各个位置处形变传感器的光路变化现象或者形变偏移量或者形变传感器耦合部受到的作用力信息中的至少一种信息判断是否存在特征信息，获得目标物质发生特异性作用过程中产生的关于形变和力的变化特征信息 和位置分布信息 ；或者
    获取单元设置有介电特征检测机构和特征信息处理器，所述特征信息处理器根据形变生物传感器作用于组织层面过程中获得的各个位置处的介电参数的变化信息判断是否存在特征信息，获得目标物质发生特异性作用过程中产生的关于介电性能的变化特征信息 和位置分布信息 ；或者
    获取单元设置有光子特征检测机构和特征信息处理器，所述特征信息处理器根据形变生物传感器作用于组织层面过程中获得的各个位置处的光的光强或频率的变化信息判断是否存在特征信息，获得目标物质发生特异性作用过程中产生的关于光的变化特征信息 和位置分布信息 。
PCT/CN2023/105772 2022-07-05 2023-07-04 组织层面单分子分辨率水平目标物质原位成像方法 WO2024008101A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210782913.8A CN115165968A (zh) 2022-07-05 2022-07-05 组织层面单分子分辨率水平目标物质原位成像方法
CN202210782913.8 2022-07-05

Publications (1)

Publication Number Publication Date
WO2024008101A1 true WO2024008101A1 (zh) 2024-01-11

Family

ID=83490932

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/105772 WO2024008101A1 (zh) 2022-07-05 2023-07-04 组织层面单分子分辨率水平目标物质原位成像方法

Country Status (2)

Country Link
CN (1) CN115165968A (zh)
WO (1) WO2024008101A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115165968A (zh) * 2022-07-05 2022-10-11 华南理工大学 组织层面单分子分辨率水平目标物质原位成像方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102401843A (zh) * 2010-09-10 2012-04-04 中国科学院沈阳自动化研究所 一种基于原子力显微术的单克隆抗体靶向药疗效检测方法
CN103197102A (zh) * 2013-03-08 2013-07-10 西南大学 基于多功能探针的单细胞/单分子成像光/电综合测试仪
CN103792393A (zh) * 2014-01-22 2014-05-14 中国科学院长春应用化学研究所 基于原子力显微镜的单粒子或单分子示踪装置及示踪方法
KR101737450B1 (ko) * 2015-12-09 2017-05-18 포항공과대학교 산학협력단 단일 세포 내 마이크로 rna의 고해상도 시각화 방법
CN111693695A (zh) * 2020-05-15 2020-09-22 华南理工大学 抗原与抗体蛋白结合动态作用过程信号采集及处理方法
CN115165968A (zh) * 2022-07-05 2022-10-11 华南理工大学 组织层面单分子分辨率水平目标物质原位成像方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102401843A (zh) * 2010-09-10 2012-04-04 中国科学院沈阳自动化研究所 一种基于原子力显微术的单克隆抗体靶向药疗效检测方法
CN103197102A (zh) * 2013-03-08 2013-07-10 西南大学 基于多功能探针的单细胞/单分子成像光/电综合测试仪
CN103792393A (zh) * 2014-01-22 2014-05-14 中国科学院长春应用化学研究所 基于原子力显微镜的单粒子或单分子示踪装置及示踪方法
KR101737450B1 (ko) * 2015-12-09 2017-05-18 포항공과대학교 산학협력단 단일 세포 내 마이크로 rna의 고해상도 시각화 방법
CN111693695A (zh) * 2020-05-15 2020-09-22 华南理工大学 抗原与抗体蛋白结合动态作用过程信号采集及处理方法
CN115165968A (zh) * 2022-07-05 2022-10-11 华南理工大学 组织层面单分子分辨率水平目标物质原位成像方法

Also Published As

Publication number Publication date
CN115165968A (zh) 2022-10-11

Similar Documents

Publication Publication Date Title
US11022598B2 (en) Devices and methods for sample analysis
US10571465B2 (en) Plasmonic substrates for metal-enhanced fluorescence based sensing, imaging and assays for cells
Li et al. Simultaneous electrochemical immunoassay of three liver cancer biomarkers using distinguishable redox probes as signal tags and gold nanoparticles coated carbon nanotubes as signal enhancers
JP6260541B2 (ja) 夾雑物の影響を低減する免疫測定法
WO2024008101A1 (zh) 组织层面单分子分辨率水平目标物质原位成像方法
Wang et al. Probing single molecule binding and free energy profile with plasmonic imaging of nanoparticles
CN111334282B (zh) Pth稀土检测试剂盒及检测卡及其微球及制备与检测方法
KR20050100367A (ko) 소용적 샘플 내의 분자 분석용 방법 및 장치
AU2010206657A1 (en) Apparatus and methods for detecting inflammation using quantum dots
CN103116023A (zh) 用于检测肿瘤标志物的电化学发光免疫传感器及其制备方法和应用
US11480541B2 (en) Optical imaging of single molecule size, charge, mobility, binding and conformational change
US20110275092A1 (en) Method for detecting biological markers by an atomic force microscope
CN107561054A (zh) 金银双金属三维有序大孔结构作为sers基底用于心肾综合征多种蛋白质同时检测
JP5958339B2 (ja) 近接場増強蛍光センサチップ
JP2020523555A (ja) 試料中における細胞外小胞の検出方法
CN112074740A (zh) 成像测定法
Vo‐Dinh et al. Single‐cell monitoring using fiberoptic nanosensors
JP4910132B2 (ja) 表面電荷量計測装置及び表面電荷量計測方法
WO2015038967A1 (en) Universal bio diagnostic, drug delivery device and marker for correlated optical and electron microscopy
JP5433698B2 (ja) 固体生物学的物体の分析のための方法
US11499967B2 (en) Specific protein marker and method for identifying the statistic distribution of protein stoichiometry
CN108693144B (zh) 基于sprm技术进行复用性单细胞蛋白质组学监测方法
CN111693695A (zh) 抗原与抗体蛋白结合动态作用过程信号采集及处理方法
AU2004230714B8 (en) Molecular detection method, molecular counting method, molecular localization detection method, and molecular detection system used therefor
KR102267198B1 (ko) 업컨버전 나노입자를 이용한 생체물질 분석용 광학 바이오센서, 및 이를 이용한 생체물질 정량 분석 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23834868

Country of ref document: EP

Kind code of ref document: A1