Patent Publication Number: US-2023160795-A1

Title: Isolation chip assembly

Description:
FIELD 
     The subject matter herein generally relates to biotechnology, and more particularly, to an isolation chip assembly for isolation and purification of target particles from a liquid sample. 
     BACKGROUND 
     Exosomes are small vesicles with a double phospholipid membrane structure, and has a diameter of 30-150 nm continuously secreted by living cells. As a carrier of intercellular communication, the exosomes carry specific components such as proteins, nucleic acids, and metabolic small molecules from mother cells. A large number of studies have shown that the exosomes are involved in a variety of events in tumor development, including immune escape, angiogenesis, tumor metastasis, tumor drug resistance, and so on. Exocrine can be released by cancer cells earlier and continuously and enter the patient’s blood circulation system. The double phospholipid membrane structure can effectively protect the carried proteins and encapsulated nucleic acids. The exosomes widely and stably exist in a variety of clinical samples, including blood, urine, ascites, tissue fluid, tears, saliva, and cerebrospinal fluid. There are many exosomes in blood and urine, so it is easy to obtain clinical samples. Therefore, the exosomes are considered to be the key research objects in the field of in vitro diagnosis and clinical detection of tumors, and are expected to play a great clinical value in early diagnosis of tumors, evaluation of tumor metastasis and recurrence, evaluation of tumor heterogeneity, dynamic detection of tumor occurrence, development and curative effect, detection of drug-resistant mutations, personalized drug use, and so on. 
     At present, a main obstacle to the clinical application of the exosomes is that the filtration membrane is blocked during an isolation process, and the isolation has low flux and low purity. 
     SUMMARY 
     Therefore, an isolation chip assembly for overcome the above shortcomings is needed. 
     The present disclosure provides an isolation chip assembly for isolation and purification of target particles from a liquid sample. The isolation chip assembly includes an isolation chip, first oscillators, and second oscillators. The isolation chip includes a sample reservoir, and a first filtration membrane and a second filtration membrane disposed at opposite sides of the sample reservoir. Sizes of pores of each of the first filtration membrane and the second filtration membrane are smaller than sizes of the target particles. The isolation chip further includes a first chamber and a second chamber, the first chamber is connected to the sample reservoir through the first filtration membrane, the second chamber is connected to the sample reservoir through the second filtration membrane. The first oscillators are mounted on the first filtration membrane and the second filtration membrane, and can generate a first oscillation wave when operating. The second oscillators are mounted on outer surfaces of the first chamber and the second chamber, and can generate a second oscillation wave when operating. A frequency of the first oscillation wave is greater than a frequency of the second oscillation wave, an amplitude of the first oscillation wave is less than an amplitude of the second oscillation wave. 
     In some possible implementations, the frequency of the first oscillation wave is 5000 Hz to 8000 Hz; the frequency of the second oscillation wave is 100 Hz to 500 Hz. 
     In some possible implementations, the frequency of the first oscillation wave is equal to a resonance frequency of the first filtration membrane or the second filtration membrane. 
     In some possible implementations, the first oscillators and the second oscillators are located on a same horizontal plane. 
     In some possible implementations, the isolation chip further includes a first side cover and a second side cover , the first side cover includes a first cover body, and a first barrier sheet and a second barrier sheet located on opposite sides of the first cover body, the first filtration membrane is fixed between the first barrier sheet and the second barrier sheet, and faces the first cover body, the first cover body, the first barrier sheet, the second barrier sheet, and the first filtration membrane cooperatively define the first chamber, the second side cover includes a second cover body, and a third barrier sheet and a fourth barrier sheet located on opposite sides of the second cover body, the third barrier sheet faces the first barrier sheet, the fourth barrier sheet faces the second barrier sheet, the second filtration membrane is fixed between the third barrier sheet and the fourth barrier sheet, and faces the second cover body, the second cover body, the third barrier sheet, the fourth barrier sheet, and the second filtration membrane cooperatively define the second chamber; the sample reservoir is disposed between the first filtration membrane and the second filtration membrane. 
     In some possible implementations, the second oscillators are fixed to outer surfaces of the first cover body and the second cover body. 
     In some possible implementations, the first chamber defines a first outlet that connects the first chamber to an ambient environment; the second chamber defines a second outlet that connects the second chamber to the ambient environment. 
     In some possible implementations, each of the first oscillators is a harmonic oscillator. 
     In some possible implementations, each of the second oscillators is a vibrating motor. 
     Compared to the existing device, the first oscillators of the present disclosure can transmit the first oscillation wave to the first filtration membrane and the second filtration membrane, so that the target particles adsorbed in the pores of the filtration membranes can be quickly separated from the pores of the filtration membranes and resuspended in the liquid sample. The second oscillators can transmit the second oscillation wave, and the second oscillation wave and the first oscillation wave can cooperatively disturb the liquid sample and the filtration membranes to generate an acoustic streaming, which prevents the target particles from clogging the pores or from gathering together, and improves the isolation and purification efficiency. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    is a diagrammatic view of an embodiment of an isolation chip assembly according to the present disclosure. 
         FIG.  2    is a diagrammatic view showing a negative pressure applied on the isolation chip assembly of  FIG.  1   . 
         FIG.  3    is a diagrammatic view showing a first oscillation wave and a second oscillation wave applied to the isolation chip assembly of  FIG.  1   . 
         FIG.  4    is a block diagram of an embodiment of an isolation device according to the present disclosure. 
         FIG.  5    is a relationship between volumes of exosomes obtained by Example and Comparative examples 1-4 and purification times. 
         FIG.  6    is a scanning electron micrograph (SEM) of exosomes obtained by Example of the present disclosure. 
         FIG.  7    is a diagram of concentrations of exosomes obtained from urine samples of different volumes and concentrations of exosomes. 
         FIG.  8    is a diagram of concentrations of exosomes obtained from different types of liquid samples. 
         FIG.  9    is Western blot analysis of exosomes obtained by Example and Comparative example 5. 
         FIG.  10    is a diagram showing a comparison between Example 5 and Comparative Example 5 in three dimensions including purification time, exosomes yield, and purity of exosomes. 
     
    
    
     Main description of component symbols: Isolation chip assembly  1 ; isolation chip  10 ; first side cover  11 ; second side cover  12 ; sample reservoir  13 ; first filtration membrane  14 ; first chamber  15 ; second filtration membrane  16 ; second chamber  17 ; sample adding chamber  18 ; first oscillator  20 ; second oscillator  30 ; frequency converting module  40 ; vacuum system  50 ; controller  60 ; isolation device  100 ; first cover body  110 ; first barrier sheet  111 ; second barrier sheet  112 ; second cover body  120 ; third barrier sheet  121 ; fourth barrier sheet  122 ; sample injection inlet  131 ; first outlet  152 ; second outlet  172 ; frequency converter  410 ; valve  420 ; first vacuum pump  510 ; second vacuum pump  520 . 
     Implementations of the present disclosure will now be described, by way of embodiments only, with reference to the attached figures. 
     DETAILED DESCRIPTION 
     The technical solution of this application will be described below in combination with embodiments and examples of this application. It should be noted that when a unit is described as “connected to” another unit, the unit can either be directly connected to the another unit, or an intermediate unit may exist therebetween. When a unit is described as “disposed on” another unit, the unit may be set directly on another unit, or an intermediate unit may exist therebetween. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those skilled in the art. The names of components or devices used in the application are for describing the specific embodiments, but are not intended to limit the scope of the application. 
       FIG.  1    illustrates an embodiment of an isolation chip assembly  1  adapted for isolation and purification of different particles from a liquid sample, so as to obtain target particles of particularly sizes. The liquid sample can be a bioliquid such as plasma, serum, saliva, urine, and lavage. The target particles can be biological cells such as circulating tumor cells (CTCs) or exosomes. The isolation chip assembly  1  includes an isolation chip  10 , a first oscillator  20 , and a second oscillator  30 . 
     The isolation chip  10  includes a sample reservoir  13 , and a first filtration membrane  14  and a second filtration membrane  16  at opposite sides of the sample reservoir  13 . The sizes of the pores of the first filtration membrane  14  and the pores of the second filtration membrane  16  are smaller than the size of the target particles. 
     Furthermore, the isolation chip  10  further includes a first chamber  15  and a second chamber  17 . The first chamber  105  is connected to the sample reservoir  13  by the first filtration membrane  14 . The first chamber  15  includes a first outlet  152  that connects the first chamber  15  to an ambient environment. The second chamber  17  is connected to the sample reservoir  13  by the second filtration membrane  16 . The second chamber  17  includes a second outlet  172  that connects the second chamber  17  to the ambient environment. The first chamber  15  and the second chamber  17  can be positioned at the opposite sides of the sample reservoir  13 . 
     In use, the liquid sample is added to the sample reservoir  13 . Each of the first outlet  152  and the second outlet  172  is connected to a vacuum system  50  (shown in  FIG.  4   ). When the vacuum system  50  evacuates the first chamber  15  through the first outlet  152 , a negative pressure is generated in the first chamber  15 . Under the negative pressure in the first chamber  15 , compositions in the liquid sample that are smaller than the pores of the first filtration membrane  14  (including small particles and liquid) can flow towards the first filtration membrane  14  and then enter the first chamber  15  through the first filtration membrane  14 . When the vacuum system  50  evacuates the second chamber  17  through the second outlet  172 , a negative pressure is generated in the second chamber  17 . Under the negative pressure in the second chamber  17 , compositions in the liquid sample that are smaller than the pores of the second filtration membrane  16  can flow towards the second filtration membrane  16  and then enter the second chamber  17  through the second filtration membrane  16 . At the same time, the back flow of the liquid sample adjacent to the first filtration membrane  14  prevents any composition from accumulating in the pores of the first filtration membrane  14 . Thus, clogging of the first filtration membrane  14  can be avoided. Since the negative pressure is alternately applied in the first chamber  15  and the second chamber  17 , the compositions in the liquid sample can alternately flow through the first filtration membrane  14  and the second filtration membrane  16 . This leaves the particles that are larger than the pores of the first filtration membrane  14  and the second filtration membrane  16  (that is, the target particles) in the sample reservoir  13 . The design of the isolation chip  10  makes the components adsorbed on the first filtration membrane  14  and the second filtration membrane  16  easy to be separated therefrom under the alternated negative pressures, which can effectively prevent the pores of the filter membrane from being blocked. 
     Referring to  FIG.  2   , in an embodiment, the negative pressure (NP) alternating between the first chamber  15  and the second chamber  17  is caused by trapezoidal wave shaped pulse signals. The rectangular wave shaped pulse signals have an amplitude of 10 V pp , and a frequency of 5000 Hz to 7000 Hz. The trapezoidal wave shaped pulse signal can avoid damage to the first filtration membrane  14  and the second filtration membrane  16  caused by sudden change of direction of the negative pressure. Since a high protein concentration is included in a plasma sample, to further avoid the clogging of the filtration membranes, an air pressure (AP) can be applied to one chamber when the negative pressure is applied to the other chamber, thereby improving the back flow at the filtration membranes. 
     Referring to  FIG.  1   , two first oscillators  20  are included. One of the first oscillators  20  is mounted on a surface of the first filtration membrane  14  away from the second filtration membrane  16 . The other one of the first oscillators  20  is mounted on a surface of the second filtration membrane  16  away from the first filtration membrane  14 . The first oscillators  20  can generate a first horizontal oscillation wave when operating, and then transmit the first oscillation wave to the first filtration membrane  14  and the second filtration membrane  16  to drive the first filtration membrane  14  and the second filtration membrane  16  to vibrate at a high frequency. Therefore, the target particles adsorbed in the pores of the filtration membranes can be quickly separated from the pores of the filtration membranes and resuspended in the liquid sample, thereby further avoiding the clogging of the filtration membranes and obtaining an efficient isolation. 
     Two second oscillators  30  are included. One of the second oscillators  30  is mounted on an outer surface of the first chamber  15 . The other one of the second oscillators  30  is mounted on an outer surface of the second chamber  17 . The second oscillators  30  can generate a second horizontal oscillation wave when operating. Referring to  FIG.  3   , the frequency of the first oscillation wave is greater than the frequency of the second oscillation wave, but the amplitude of the first oscillation wave is less than the amplitude of the second oscillation wave. The second oscillation wave is transmitted to the whole isolation chip  10  through the first chamber  15  and the second chamber  17 , thus the isolation chip  10  can vibrate at a low frequency. The first oscillation wave and the second oscillation wave can cooperatively disturb the liquid sample and the filtration membranes to generate an acoustic streaming, which prevents the target particles from clogging the pores or from gathering together, and improves the isolation and purification efficiency. In at least one embodiment, the first oscillator  20  may be a harmonic oscillator, and the second oscillator  30  may be a vibrating motor. 
     In at least one embodiment, the frequency of the first oscillation wave is 5000 Hz to 8000 Hz. The frequency of the second oscillation wave is 100 Hz to 500 Hz. Neither the first oscillation wave nor the second oscillation wave at such frequency will cause damage to the target particles. In at least one embodiment, the frequency of the first oscillation wave can be approximately the same as the resonance frequency of the first filtration membrane  14  or the second filtration membrane  16 . Thus, the first filtration membrane  14  or the second filtration membrane  16  can vibrate with a larger amplitude, thereby causing the target particles adsorbed on the filtration membranes to be separated from the filtration membranes more quickly. 
     In at least one embodiment, the first oscillators  20  and the second oscillators  30  are located on a same horizontal plane. That is, the first oscillation wave and the second oscillation wave are towards a same direction, so that the first oscillation wave and the second oscillation wave can be superimposed on each other to form a coordinated oscillation wave. 
     In at least one embodiment, the isolation chip  10  further includes a first side cover  11  and a second side cover  12 . The first side cover  11  includes a first cover body  110 , and a first barrier sheet  111  and a second barrier sheet  112  located on opposite sides of the first cover body  110 . The first filtration membrane  14  is fixed between the first barrier sheet  111  and the second barrier sheet  112 , and faces the first cover body  110 . The first cover body  110 , the first barrier sheet  111 , the second barrier sheet  112 , and the first filtration membrane  14  cooperatively define the first chamber  15 . The second side cover  12  includes a second cover body  120 , and a third barrier sheet  121  and a fourth barrier sheet  122  located on opposite sides of the second cover body  120 . The third barrier sheet  121  faces the first barrier sheet  111 . The fourth barrier sheet  122  faces the second barrier sheet  112 . The second filtration membrane  16  is fixed between the third barrier sheet  121  and the fourth barrier sheet  122 , and faces the second cover body  120 . The second cover body  120 , the third barrier sheet  121 , the fourth barrier sheet  122 , and the second filtration membrane  16  cooperatively define the second chamber  17 . The sample reservoir  13  is disposed between the first filtration membrane  14  and the second filtration membrane  16 . The second oscillator  30  is fixed to an outer surface of the first cover body  110  or the second cover body  120 . 
     Furthermore, the first barrier sheet  111  and the third barrier sheet  121  are spaced from each other to define a sample injection inlet  131  that communicates with the sample reservoir  13 . The isolation chip  10  further includes a sample adding chamber  18  that communicates with the sample reservoir  13  through the sample injection inlet  131 . During use, the liquid sample is added to the sample adding chamber  18 , and the sample injection inlet  131  allows the liquid sample in the sample adding chamber  18  to flow out and enter the sample reservoir  13 . 
       FIG.  4    illustrates an embodiment of an isolation device  100  including the isolation chip assembly  1 , a vacuum system  50 , a frequency converting module  40 , and a controller  60 . 
     The vacuum system  50  generates the negative pressure in the first chamber  15  and the second chamber  17  of the isolation chip assembly  1  alternately. In at least one embodiment, the vacuum system  50  includes a first vacuum pump  510  and a second vacuum pump  520 . The first vacuum pump  510  is connected to tire first outlet  152  of the isolation chip  10 . The second vacuum pump  520  is connected to the second outlet  172  of the isolation chip  10 . 
     The frequency converting module  40  is electrically connected to the vacuum system  50 , and provides electric power to the vacuum system  50 . In an embodiment, the frequency converting module  40  includes a frequency converter  410  and a valve  420  connected to the frequency converter  410 . The valve  420  can be an electromagnetic valve or a rotary valve. The valve  420  is alternately switched to connect one of the first vacuum pump  510  and the second vacuum pump  520 , to cause the vacuum system  50  to alternately apply the negative pressure in the first chamber  15  and the second chamber  17 . That is, when the valve  420  connects to the first vacuum pump  510 , the frequency converter  410  controls the first vacuum pump  510  to generate the negative pressure in the first chamber  15 . The compositions that have sizes smaller than the size of the pores of the first filtration membrane  14  can pass through the first filtration membrane  14  under the negative pressure. Then, the frequency converter  410  controls the first vacuum pump  510  to stop operating, and the valve  420  is switched to connect to the second vacuum pump  520 . The frequency converter  410  controls the second vacuum pump  520  to apply the negative pressure in the second chamber  17 . The compositions that have sizes smaller than the size of the pores of the second filtration membrane  16  can pass through the second filtration membrane  16  under the negative pressure. Then, the frequency converter  410  controls the second vacuum pump  520  to stop operating. The above procedures are repeated until complete isolation is achieved. 
     The controller  60  controls the first oscillator  20  and the second oscillator  30  to operate when the first chamber  15  is stopped evacuated. Thus, the first oscillation wave and the second oscillation wave are generated. The controller  60  further controls the first oscillator  20  and the second oscillator  30  to operate when the second chamber  17  is stopped evacuated. The controller  60  can be electrically connected to the first vacuum pump  510  and the second vacuum pump  520 . When the first vacuum pump  510  or the second vacuum pump  520  stops operating, the controller  60  determines that the first vacuum pump  510  stops evacuating the first chamber  15  or the second vacuum pump  520  stops evacuating the second chamber  17 . Then, the controller  60  informs the first oscillator  20  and the second oscillator  30  to start operating. 
     An embodiment of an isolation method of isolating target particles from a liquid sample, which is executed by the isolation chip assembly  1 , is also provided. The method includes the following steps. 
     S 1 , the isolation chip assembly  1  is provided, and the liquid sample is added to the sample reservoir  13  of the isolation chip assembly  1 . 
     S 2 , the first chamber  15  is evacuated through the first outlet  152  to generate the negative pressure in the first chamber  15 . 
     In at least one embodiment, before evacuating the first chamber  15 , the first outlet  152  and the second outlet  172  are connected to the vacuum system  50 . Then, the vacuum system  50  evacuates the first chamber  15  through the first outlet  152 , to cause the compositions having sizes that are smaller than sizes of the pores of the first filtration membrane  14  to enter the first chamber  15  through the first filtration membrane  14 . 
     S 3 , vacuuming of the first chamber  15  is stopped, and the first oscillator  20  and the second oscillator  30  operate to generate the first oscillation wave and the second oscillation wave. At the same time, the second chamber  17  is evacuated through the second outlet  172  to generate the negative pressure in the second chamber  17 . 
     The first oscillation wave drives the first filtration membrane  14  to vibrate at a high frequency. Therefore, the target particles adsorbed in the pores of the filtration membranes can be quickly separated from the pores of the filtration membranes and resuspended in the liquid sample. The second chamber  17  can prevent the target particles from gathering together. At the same time, since the negative pressure is generated in the second chamber  17 , the compositions adhered on the first filtration membrane  14 , which have sizes smaller than the size of the pores of the second filtration membrane  16 , can return to the sample reservoir  13  together with the flows of the fluid, and further move towards the second chamber  17  through the second filtration membrane  16 . 
     S 4 , vacuuming of the second chamber  17  is stopped, and the first oscillator  20  and the second oscillator  30  operate. 
     Then, the steps S 2  to S 4  can be repeated for several times, so that the components smaller than the pores of the filtration membranes in the liquid sample are removed, and the components larger than the pores of the filtration membranes are remained in the sample reservoir  13 , so as to achieve better isolation and purification effect. 
     The present application will be described in detail in combination with specific examples and comparative examples. 
     EXAMPLE 
     The exosomes are isolated and purified from a urine sample of 2 mL by the isolation chip assembly of the present disclosure. The frequency of the first oscillator is 6250 Hz (approximately the same as the resonance frequency of the filtration membrane), and the frequency of the second oscillator is 200 Hz. 
     Comparative Example 1 
     Different from Example 1, the exosomes are isolated and purified from a urine sample of 2 mL by the isolation chip. The first oscillator and the second oscillator are not included in the isolation chip. 
     Comparative Example 2 
     Different from Example 1, the exosomes are isolated and purified from a urine sample of 2 mL by the isolation chip. The first oscillator is not included in the isolation chip. 
     Comparative Example 3 
     Different from Example 1, the exosomes are isolated and purified from a urine sample of 2 mL by the isolation chip. The second oscillator is not included in the isolation chip. 
     Comparative Example 4 
     The exosomes are isolated and purified from a urine sample of 2 mL by dead-end filtration. The dead-end filtration uses the same filtration membrane as the isolation chip, but the liquid sample is placed upstream of the filtration membrane. Under the function of pressure difference, components that have sizes smaller than the size of the pores of the filtration membrane are allowed to pass through the filtration membrane. 
     The concentrations of the exosomes obtained in Example and Comparative Examples 1-4 are measured, and the results are recorded in  FIG.  5   . As shown in  FIG.  5   , the exosomes of nearly 30 µm can be isolated from the liquid sample within 10 minutes in Example, and the isolation efficiency is much higher than that in the Comparative examples 1-4. The exosomes obtained in the Example are further subjected to a transmission electron microscope test, and the test result is shown in  FIG.  6    (the scale is 250 nm).  FIG.  6    shows that the exosomes have a particle size of 50 nm to 200 nm, which is consistent with the theoretical size of the exosomes. The exosomes are round or cup-shaped, which have a high integrity. 
     Furthermore, the same isolation chip assembly is used to repeat the isolation and purification of exosomes from four urine samples. The four urine samples have different concentrations of exosomes. Each of the four urine samples has a volume of 1 mL to 20 mL. Then, an ultraviolet-visible spectrophotometer is used to test the concentrations of proteins in the exosomes, and the results are recorded in  FIG.  7   . As shown in  FIG.  7   , the concentrations of exosomes linearly increase with the increase of the volume of the urine samples, indicating that when the liquid samples having different volumes or when the liquid samples having exosomes with different concentrations are tested, the isolation chip assembly has a high structural stability during the isolation and the purification of the exosomes. In addition, the same isolation chip assembly is repeatedly used for the isolation and the purification of the exosomes from a urine sample of 10 mL. After twenty times, the coefficient of variation (CV) between the concentrations of exosomes is less than 9.9%, indicating that the isolation chip assembly has a high repeatability during the isolation and the purification of the exosomes. 
     Furthermore, the isolation chip assembly is also used to isolate and purify exosomes from other liquid samples, including plasma, cell culture medium, tear, saliva, and cerebrospinal fluid (CSF). As shown in  FIG.  8   , higher concentrations of exosomes are obtained from these liquid samples, indicating that the isolation chip assembly is suitable for the isolation and the purification of exosomes from various liquid samples. Moreover, the size of the exosomes is also in the range of 50 nm to 200 nm. 
     Comparative Example 5 
     Existing isolation and purification methods, such as ultracentrifugation (UC), polyethylene glycol (PEG) precipitation, phosphatidylserine (PS) affinity, size exclusion chromatography (SEC), and membrane affinity (MA), are used to isolate exosomes from the same urine sample. 
     Western blotting is used to test protein markers including ALIX, CD63, TSG101, and CD81 in the exosomes obtained by Example and Comparative example 5. Uromodulin (UMOD) is a protein with a highest concentration in the urine samples, which can be used to characterize the purity of the exosomes. As shown in  FIG.  9   , compared with the exosomes isolated by the existing isolation and purification methods, four protein markers can be detected in the exosomes isolated and purified by the isolation chip assembly (denoted as EXODUS in  FIG.  9   ), indicating that the purification yield is high. The exosomes do not adsorb a large amount of uromodulin, indicating that the purification accuracy is high. 
     The purification time, the exosomes yield, and the purity of exosomes are three dimensions to characterize different isolation and purification methods. As shown in  FIG.  10   , compared to the existing isolation and purification methods in Comparative Example 5, the isolation chip assembly (denoted as EXODUS in  FIG.  10   ) requires a shorter purification time (reduced by 95%), a higher exosomes yield (increased by 526%), and a higher purity of exosomes (increased by 259%), indicating that the isolation chip assembly is more competitive than the existing isolation and purification methods. 
     The embodiments shown and described above are only examples. Therefore, many commonly-known features and details are neither shown nor described. Even though numerous characteristics and advantages of the present technology have been set forth in the foregoing description, together with details of the structure and function of the present disclosure, the disclosure is illustrative only, and changes may be made in the detail, including in matters of shape, size, and arrangement of the parts within the principles of the present disclosure, up to and including the full extent established by the broad general meaning of the terms used in the claims. It will, therefore, be appreciated that the embodiments described above may be modified within the scope of the claims.