Detailed Description
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be further noted that, for the convenience of description, only some but not all of the relevant aspects of the present invention are shown in the drawings.
The technical scheme of the invention is realized on the basis of a microfluidic technology and a Coulter principle. The microfluidic technology is a technology for accurately controlling and controlling microscale fluid, particularly a submicron structure, in the fields of researching fluid behaviors on scale and mesoscale or nano-scale, related design and application, and comprising subjects such as physics, chemistry, micromachining, biotechnology and the like. The development of microfluidic technology has made it possible to determine and analyze particles in fluids at the micro-scale. The coulter principle, also known as a resistance method, an electric pulse method or an electric induction zone technology, means that when particles suspended in electrolyte pass through a small-hole tube along with the electrolyte, namely a micro-channel, the same volume of electrolyte is replaced, and in a constant current designed circuit, the resistance between two electrodes inside and outside the small-hole tube is instantaneously changed to generate potential pulses. The magnitude and number of pulse signals is proportional to the size and number of particles. Therefore, the electrolyte, namely the particles in the liquid can be measured and analyzed according to the size and the times of the pulse signals. The present invention will be further described with reference to the accompanying drawings and the detailed description.
FIG. 1 is a schematic view of an apparatus for detecting and analyzing particles in a liquid according to a first embodiment of the present invention; preferably, in this embodiment, the technical solution of the present invention is described by taking the measurement and analysis of single particles and double particles in the liquid as an example. As shown in fig. 1, the apparatus includes:
a sample cell 101, a reagent detector 102, a sample valve 103 provided with a water conduit (not shown in the figure), a mixing cell 105 provided with at least one reagent cell 104, a bubble eliminator 106, a test valve 107 and a sensor chip 108.
The sample injection cell 101, the mixing cell 105 and the bubble eliminator 106 are respectively connected with the sample injection valve 103, and the opening direction of the sample injection valve 103 is adjusted to respectively realize the conduction of the sample injection cell 101, the mixing cell 105 and the bubble eliminator 106 with the sample injection valve 103.
The bubble eliminator 106 is used for eliminating bubbles of the reagent sample in the injection valve 103 when the sample eliminator is communicated with the injection valve 103. Further, the bubble eliminator is also used to eliminate bubbles in the entire tube while eliminating bubbles in the reagent sample. After eliminating the bubble in the whole pipe, promoted the velocity of flow of liquid in the pipe, promoted test speed and measurement accuracy.
Preferably, the bubble eliminator is adopted as the waste liquid pool 106 in the embodiment, a hydrophobic breathable film is arranged, and when the waste liquid pool 106 is communicated with the sample injection valve 103, the hydrophobic breathable film is used for applying an external power source to the waste liquid pool 106, so that bubbles in the sample injection valve 103 are removed through air pressure.
The reagent detector 102 is connected to the sample injection valve 103, and is configured to adjust an opening direction of the sample injection valve 103 after detecting that the sample injection valve 103 is filled with the reagent sample, and discharge the reagent sample in a water conduit of the sample injection valve 103 into the mixing tank 105. The reagent detector 102 may take various forms to detect whether the reagent sample is filled, and is preferably an impedance detector that detects the filled position of the reagent sample by means of an impedance value.
The reagent reservoir 104 is used to add reagent in the reagent reservoir 104 to the mixing reservoir 105. At different stages of testing and analysis, different reagent reservoirs 104 may be connected as desired to introduce different reagents.
The mixing cell 105 and the sensor chip 108 are connected to the test valve 107, respectively, and the sensor chip 108 is conducted to the mixing cell 105 by adjusting the opening direction of the test valve 107, for measuring and analyzing the reagent sample flowing through the sensor chip 108.
The device for determining and analyzing particles in liquid disclosed by the embodiment of the invention can eliminate bubbles of the sample injection valve reagent sample through the bubble eliminator, so that the accuracy of quantifying the reagent sample in the aqueduct is improved, and the accuracy of determination and analysis is improved because the quantified reagent sample is a reference for analysis and determination. Furthermore, the technical scheme of the embodiment of the invention is provided with the sample injection valve and the test valve which are flexibly connected with different devices, and the flow direction of the reagent sample can be controlled, so that the structure is compact, the volume of the device can be reduced, and the device is convenient to carry.
In this embodiment, the sample inlet well 101, or sample inlet structure, is a passage for a reagent sample to enter the device, and may be a small hole that is matched to a needle that obtains the sample, as well as other structures that add reagent samples to the device as will be understood by those skilled in the art. Preferably a capillary, pipette or syringe or other sample dispensing structure, for driving the reagent sample into the sample reservoir by capillary action or external force.
The sample injection valve 103 is a device for controlling the device to quantify the reagent sample and is communicated with the sample injection cell 101 and the waste liquid cell 106. The sample injection valve 103 is provided with a water conduit, and according to a specific application mode and an application scene, the water conduit is provided with a corresponding accommodating volume, and the corresponding accommodating volume provides a reference basis for determining a measurement result. The water conduit is disposed in the injection valve 103 for quantifying a predetermined amount of the reagent sample after the injection valve 103 is filled with the reagent sample. The specific water conduit may share a conduit with the sample injection valve 103, or may be a conduit independent of the conduit in the sample injection valve 103. Furthermore, when the opening direction of the sample injection valve 103 is adjusted to communicate the sample injection cell 101 with the waste liquid cell 106, the sample injection valve is used to discharge the redundant reagent sample in the sample injection cell 101 or the sample injection valve 103 into the waste liquid cell 106. Therefore, the waste liquid tank 106 can have multiple functions, such as storing waste liquid, driving liquid to flow, and the like, thereby effectively simplifying the structure of the device. Preferably, the waste liquid pool 106 is provided with a hydrophobic and breathable membrane, and the waste liquid pool 106 is connected with the mixing pool 105 through the sensor chip 108, so that an external power source acts on the hydrophobic and breathable membrane of the waste liquid pool 106 to drive the reagent sample in the mixing pool 105 to flow into the sensor chip 108 through air pressure.
Preferably, in this embodiment, the conduit in the sample injection valve 103 and the water conduit disposed in the sample injection valve 103 share one conduit.
The waste liquid tank 106 is used for receiving redundant reagent samples or reagent samples to be processed in the sample injection tank 101, the sample injection valve 103 and the mixing tank 105, and is used for receiving reagent samples or processing reagent samples after measurement and analysis. The reagent sample or process reagent sample in the mixing cell 105 can be driven into the sensor chip 108 by creating pressure by pumping gas at the waste cell 106 port. Meanwhile, by applying an external power source to the waste liquid tank 106, when the opening direction of the injection valve 103 is adjusted, the excess reagent sample in the injection valve 103 or the injection tank can be discharged into the waste liquid tank 106, and when the opening direction of the test valve 107 is adjusted, the reagent sample in the mixing tank 105 can be discharged into the waste liquid tank 106.
FIG. 2 is a schematic view of a waste liquid tank according to a first embodiment of the present invention; as shown in fig. 2, the waste liquid tank 106 includes: an opening 201 and a hydrophobic and breathable membrane 202, and waste liquid 203 exists in the waste liquid pool 106. Wherein the hydrophobic gas permeable membrane 202 is a thin membrane through which only small molecules of gas can pass, but not large molecules of liquid. The material of the membrane may be polytetrafluoroethylene, a polytetrafluoroethylene derivative, a polytetrafluoroethylene-like material, or the like.
In this embodiment, the waste liquid tank 106 is provided with a hydrophobic gas-permeable membrane 202; when an external power source is applied to the waste liquid cell 106, it can be used to remove air bubbles in the sample injection valve 103 or to drive the reagent sample in the mixing cell 105 to flow into the sensor chip 108. That is, when a test or sample injection is required or air bubbles in the test valve 103 are removed, an external power source is applied to the opening 201, so that the liquid of the device flows under the action of the external power source, and the test, sample injection or air bubble exhaust is realized.
The mixing tank 105 is a region where a reagent sample is treated according to the properties of a test reagent sample after the quantitative reagent sample is obtained. Accordingly, a corresponding number of reagent wells, for example, 2, 3, or 4 reagent wells, may be provided in connection with the mixing well 105 according to the application requirements of the reagents; the reagent solution in the corresponding reagent pool 104 is matched with the corresponding reagent, and the reagent in the reagent pool 104 and the reagent sample interact in the mixing pool, such as dilution, chemical reaction, and binding of magnetic beads, iron beads, etc., so as to realize the processing of the reagent sample. The treated reagent sample is a treated reagent sample. The reagent in the reagent pool can be any one or more of latex particles, magnetic beads, salt, antigen and antibody and the like.
Preferably, in this embodiment, the reagent pool 104 is 4 reagent pools.
The test valve 107 is a valve port for controlling the conduction of the waste liquid tank 106 and the sensor chip 108 to the mixing tank 105, and the waste liquid tank 106 and the mixing tank 105 or the sensor chip 108 and the mixing tank 105 can be correspondingly conducted by adjusting the opening direction of the test valve 107.
The sensor chip 108 is a device for measuring and analyzing particles in a reagent sample by using the coulter principle.
In this embodiment, the sensor chip 108 includes at least two electrodes, a photoresist layer, and an insulating layer; wherein
The optical adhesive layer is provided with a microchannel, the electrodes are embedded in the optical adhesive layer, and the electrodes are in an insulating state through the insulating layers between the electrodes; the electrodes have contacts in the micro-channels for measuring and analyzing particles of the reagent sample as the particles pass through the micro-channels via the contacts of the electrodes in the micro-channels. Specifically, according to the actual application mode and application scenario, a corresponding number of electrodes may be provided, for example, 2, 3, or 4 electrodes.
FIG. 3 is a schematic diagram of a sensor chip according to an embodiment of the invention; as shown in fig. 3, the sensor chip includes: a waste reservoir 301, electrodes 302, and microchannel regions 303.
Preferably, there are 4 electrodes in the microchannel region 303 in this embodiment.
FIG. 3A is an enlarged schematic view of the micro-channel region of FIG. 3 of a sensor chip according to one embodiment of the present invention; the positional relationship of the microchannel 304 and the electrode 302 is shown in fig. 3A.
Preferably, in this embodiment, the electrodes are embedded in the photoresist layer in a stacked manner.
FIG. 3B is a schematic diagram illustrating a cross-sectional structure of a sensor chip along line AA in FIG. 3 according to an embodiment of the present invention; as shown in fig. 3B, the sensor chip includes electrodes 302, micro-channels 304, a photo-resist layer 306, and an insulating layer 305. A photoresist layer 306 is disposed on the insulating layer 305 and electrodes are disposed in the insulating layer 305 and have contacts in the microchannels 304. When a sample to be tested flows through the micro-channel 304 under the action of an external power source of the waste liquid pool and contacts with the contact of the contact electrode in the micro-channel, the detection of particles in the sample flowing through the micro-channel 304 is realized.
Preferably, in this embodiment, the electrodes are embedded in the photoresist layer in a stacked manner.
Preferably, in this embodiment, the width of the microchannel is 20 micrometers to 500 micrometers.
Preferably, in this embodiment, the thickness of the optical adhesive layer is 5 micrometers to 20 micrometers.
In the present embodiment, the reagent monitor in the measurement and analysis apparatus is preferably an impedance detector 102, and two electrodes of the impedance detector 102 are connected to two ends of the sample injection valve 103 and are used for forming an electrical connection with the reagent sample after the reagent sample enters the sample injection valve 103, so as to identify whether the sample injection valve 103 is filled with the reagent sample according to a relationship between an impedance value detected by the electrodes and a set threshold. For example, when the impedance detector 102 detects that the impedance value reaches a set threshold, the reagent sample fills the test valve 103. Wherein, the set threshold value can be set correspondingly according to the specific characteristics of the test agent sample. The impedance detector 102 is configured to detect bubbles in the sample injection valve 103, for example, if the detected impedance value changes, it indicates that the bubbles block the electrical connections of the electrodes, or the resistance between the electrical connections is changed, so as to determine that the bubbles exist. When the impedance detector 102 detects that bubbles exist in the sample injection valve 103, the bubbles in the sample injection valve are discharged by applying an external power source at the waste liquid pool 106, so that the accuracy of quantifying the reagent sample by a water pouring pipe in the sample injection valve 103 is improved. According to the accurate quantitative result, more accurate determination and analysis results can be obtained.
In this embodiment, after the impedance detector 102 detects that the sample injection valve is filled with the reagent sample, the opening direction of the sample injection valve is adjusted, the reagent sample in the water conduit arranged in the sample injection valve is discharged into the mixing tank, and the reagent in the reagent tank is added into the mixing tank to form a processed reagent sample; and adjusting the opening direction of the test valve to determine and analyze the processing reagent sample flowing through the sensor chip.
Specifically, in this embodiment, a reagent sample to be measured and analyzed enters the device through the sample injection cell; the sample introduction pool is connected with the sample introduction valve, and after the opening direction of the sample introduction valve is adjusted, a reagent sample enters the sample introduction valve, namely enters a water guide pipe arranged in the sample introduction valve. The impedance detector detects the object to be detected by using the resistance of the object to be detected and the reactive current. When the reagent sample enters the sample injection valve, the impedance detector connected with the sample injection valve starts to detect whether the sample injection valve is full of the reagent sample, namely whether bubbles exist in the sample injection valve reagent sample. When the sample injection valve is filled with the reagent sample, the resistance measured by the impedance detector is the minimum and the current is the maximum because the reagent sample is filled in the aqueduct of the sample injection valve. When the impedance detector detects that the sample injection valve is not filled with the reagent sample, the direction of the sample injection valve is adjusted at the moment to conduct the sample injection valve and the waste liquid pool, and then an external power source is applied to the waste liquid pool to remove air bubbles in the sample injection valve, so that the sample injection valve is filled with the reagent sample.
When the sample injection valve is filled with the reagent sample detected by the impedance detector, adjusting the direction of the sample injection valve, discharging the reagent sample in a water guide pipe arranged on the sample injection valve into the mixing tank, adding the reagent in the reagent tank, namely the preset reagent, into the mixing tank, and forming a treated reagent sample; the test valve is oriented to measure and analyze the process reagent sample flowing through the sensor chip. After the sample injection valve is adjusted, a water guide pipe arranged in the sample injection valve discharges a quantitative reagent sample into the mixing pool. The water conduit in the sample injection valve has a fixed holding volume when the device is designed, i.e. the holding volume of the water conduit is the volume of the quantitative reagent sample. After the reagent sample in the aqueduct is discharged into the mixing pool, the reagent preset in the reagent pool which is connected with the mixing pool can be added into the mixing pool according to the requirement, and the reagent sample in the mixing pool and the reagent in the reagent pool are mixed to form a treated reagent sample. After an external power source is applied to the opening of the waste liquid pool, a treatment reagent sample in the mixing pool flows through the sensor chip, so that the measurement and analysis of particles in the reagent sample are realized, and the particles after the measurement and analysis are discharged into the waste liquid pool.
More specifically, in the present embodiment, only one kind of particles is present for the liquid. When the reagent sample enters the device through the sample inlet tank and enters the sample inlet valve, the reagent sample in the sample inlet valve is filled in the aqueduct in the sample inlet valve under the detection of the impedance detector, and then the reagent sample filled in the aqueduct is discharged into the mixing tank. In order to achieve sufficient dilution of the reagent sample, only one reagent reservoir is required in the device for measuring and analyzing the single-particle liquid in this embodiment, and is used for containing water or other reagents. And adding the reagent in the reagent pool into the reagent sample discharged into the mixing pool through the water guide pipe in the mixing pool, and forming a treatment reagent sample after the action of the reagent sample and the reagent. And adjusting the direction of the test valve to enable the mixing tank and the sensor chip to be in a conducting state, further applying an external power source at the opening of the waste liquid tank, and enabling the treatment reagent sample in the mixing tank to flow through the sensor chip through the test valve under the action of the external power source. Thus, the properties of the particles in the liquid are determined and analyzed or counted by the magnitude of the current or the waveform generated by the current, which is generated by the electrode in the sensor chip and senses the particles in the processing reagent sample flowing through the microchannel.
More specifically, in this embodiment, two types of particles are required to be measured and analyzed in a liquid. As with the above-described method for quantifying particles, a predetermined amount of reagent solution is metered into the mixing tank through a water conduit in the sample injection valve. In order to measure and analyze two types of particles in a liquid, it is necessary to separate the two types of particles in the liquid. The two particles in the liquid can be separated by adding corresponding reagents into the reagent pool. For example, latex particles, magnetic beads, iron beads, etc. are added to the reagent reservoir, so that one of the particles in the reagent sample can bind to the reagent in the applied reagent solution, and thus can perform the fixing function. After a reagent solution containing two types of particles to be measured is added into a mixing pool containing the two types of particles to be measured, under the action of a corresponding reagent in a reagent pool, one type of particles in a reagent sample is fixed by the reagent in the reagent pool, the other type of particles is dissolved in the reagent sample, and the reagent sample treated by the reagent in the reagent pool is a treated reagent sample. And further applying a certain electric field or magnetic field around the mixing pool to fix the particles fixed in the treatment reagent on the inner wall of the mixing pool, applying an external power source at the opening of the waste liquid pool, and enabling the treatment reagent solution to flow through the sensor chip under the action of the external power source to realize the measurement and analysis of the unfixed particles. After the unfixed particles are measured and analyzed, a reagent pool arranged in the mixing pool is added with water or other solution to enter the mixing pool, and the magnetic field or the electric field applied to the periphery of the mixing pool is removed. Thus, the fixed particles are redissolved in water or other solution, and the external power source is applied again to the waste liquid pool, thereby measuring and analyzing the fixed particles under the driving of the external power source.
In the above aspect, the apparatus preferably further includes: and the magnetic device is matched with the sample injection valve, the mixing pool or the sensor chip and is used for adsorbing set particles in the fixed reagent sample.
According to the device for determining and analyzing the particles in the liquid, disclosed by the embodiment of the invention, the impedance detector detects the reagent sample entering the sample injection valve, whether the sample injection valve is filled with the reagent sample is determined according to the detection result, and the reagent sample in the water guide pipe of the sample injection valve is discharged into the mixing tank after the impedance detector detects that the sample injection valve is filled with the reagent sample, so that the bubble does not exist in the reagent sample in the water guide pipe in the sample injection valve, the bubble does not exist in the reagent sample in the water guide pipe, the accuracy of the quantitative reagent sample in the water guide pipe is improved, and the quantitative reagent sample is a reference for analysis and determination, and the accuracy of determination and analysis is improved. Furthermore, after the direction of the sample introduction valve is adjusted, the reagent sample in the water guide pipe enters the mixing tank, the preset reagent in the reagent tank which is connected with the mixing tank is added according to the characteristics of the reagent sample and the purpose of detection and enters the mixing tank, the reagent sample is processed, and then the processed reagent sample passes through the sensor chip, so that the determination and analysis of the reagent sample are realized. By adding the reagent in the reagent pool into the mixing pool, the diversity of reagent sample treatment is increased, and the flexibility of test is improved.
FIG. 4 is a schematic flow chart of a method for detecting and analyzing particles in a liquid according to a second embodiment of the present invention; the method of the embodiment of the present invention can be implemented by using the apparatus for measuring and analyzing particles in a liquid according to any embodiment of the present invention, and preferably, in the embodiment, the technical solution of the present invention is described by taking the measurement and analysis of CD4+ T cells in isolated blood as an example. For the sake of clarity, the related explanation or description will refer to the description in the first embodiment, and will not be repeated herein. As shown in fig. 4, the method includes:
step 401, adding a reagent sample to a sample injection valve through a sample injection cell.
In the step, a reagent sample is added into the sample inlet valve through the sample inlet pool. This may be achieved, for example, by means of a needle injection.
402, removing bubbles in the sample injection valve through a bubble eliminator communicated with the sample injection valve;
preferably, a waste liquid pool provided with a hydrophobic breathable film is adopted, and after an external power source acts on the hydrophobic breathable film of the waste liquid pool, bubbles in a sample injection valve and a pipeline are removed through the waste liquid pool and the sample injection valve which are communicated.
The step is to remove air bubbles in the system by applying an external power source at the position of the hydrophobic and breathable film of the waste liquid pool. Because the aqueduct is arranged in the sample injection valve at the same time, the step is to remove the air bubble in the aqueduct. Thereby quantifying a predetermined amount of the reagent sample.
And 403, after the reagent detector detects that the sample injection valve is filled with the reagent sample, quantifying a predetermined amount of the reagent sample through a water guide pipe in the sample injection valve.
The reagent sample is quantified after entering the sample injection valve, and the reagent sample with a fixed volume is quantified. The reagent detector is preferably implemented as an impedance detector. The method comprises the following specific steps:
detecting an impedance value through two electrodes in the impedance detector, wherein the two electrodes are electrically connected with the reagent sample in the sample injection valve;
and when the impedance value is lower than the set threshold value through comparison, the identification reagent sample is filled in the sample injection valve.
Step 404, adjusting the opening direction of the sample injection valve, and adding a quantitative predetermined amount of the reagent sample to the mixing pool.
This step is to add a predetermined amount of reagent sample to the mixing well after the predetermined amount of reagent sample has been quantified.
Step 405, adding a preset reagent in a reagent pool into a mixing pool according to the requirement;
the preset reagent refers to a reagent which is placed in a reagent pool in advance. The reagent can be preset in a corresponding selection mode according to the actual application mode or application scene. In this embodiment, the preset reagent may include any one or more of latex particles, magnetic beads, salts, antigen and antibody.
And step 406, enabling the reagent sample in the mixing pool to pass through a sensor chip according to a preset mode, so as to realize the measurement and analysis of particles in the reagent sample.
And setting a corresponding detection mode according to the property of the reagent sample to be detected. For example, for a reagent sample having only one particle, the detection can be performed directly without providing a separation. As another example, when there are two particles to be detected in the reagent sample, the method comprises the following steps:
separating particles in the reagent sample when two particles to be detected, a first particle and a second particle, are present in the reagent sample;
and enabling the first particles and the second particles to sequentially pass through the sensor chip, so that the particles in the reagent sample can be measured and analyzed.
And fixing the first particles or the second particles on the inner wall of the mixing pool by applying a magnetic field or an electric field around the mixing pool, and separating the particles in the liquid. That is, when two kinds of particles are present in the reagent sample, one of the particles is fixed to the applied preset reagent, and then one of the particles is fixed by a battery or a magnetic field. And after detecting the non-fixed particles, namely the first particles, removing the electric field or the magnetic field to realize the detection of the fixed second particles.
Specifically, in this embodiment, a vacuum power source is applied to the permeable membrane of the waste liquid tank, so that the liquid enters the sample injection valve through the sample injection tank. When the impedance monitor detects a low impedance value, the sample injection valve is filled with a reagent sample. Allowing the antigen-bound latex microspheres in the reagent reservoir to enter and simultaneously driving the selected volume of the sample to be tested in the aqueduct to flow into the mixing reservoir. After the microspheres are bound to monocytes expressing CD4+ molecules, the antigen-binding magnetic beads and solution pass from the reagent reservoir into the mixing reservoir, thereby binding to CD4+ T cells. When the washing solution enters the mixing pool from the reagent pool for the first washing, a magnetic field is applied to the mixing pool to fix the T cells combined with the magnetic beads at the bottom of the mixing pool. When the wash solution enters the mixing well from the reagent well for the second wash, the bound cells can be resuspended in the diluent. The test valve adjusts the direction so that the washed treatment reagent sample flows into the sensor chip for measurement.
The method for determining and analyzing particles in liquid disclosed by the embodiment of the invention comprises the steps that an impedance detector detects a reagent sample entering a sample injection valve, whether the sample injection valve is filled with the reagent sample is determined according to a detection result, and the reagent sample in a water guide pipe of the sample injection valve is discharged into a mixing pool after the impedance detector detects that the sample injection valve is filled with the reagent sample, so that the reagent sample in the water guide pipe in the sample injection valve does not have bubbles because the sample injection valve does not have bubbles, the reagent sample in the water guide pipe does not have bubbles, the accuracy of quantifying the reagent sample in the water guide pipe is improved, and the quantifying reagent sample is a reference for analysis and determination, and the accuracy of determination and analysis is improved. Furthermore, after the direction of the sample introduction valve is adjusted, the reagent sample in the water guide pipe enters the mixing tank, the preset reagent in the reagent tank which is connected with the mixing tank is added according to the characteristics of the reagent sample and the purpose of detection and enters the mixing tank, the reagent sample is processed, and then the processed reagent sample passes through the sensor chip, so that the determination and analysis of the reagent sample are realized.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.