Abstract:
A disk-based fluid sample separation device including at least one air vent forming a part of a flow channel pattern on a microfluidic disk is disclosed. The fluid sample separation device is provided with an air vent sealing cover having at least through hole and is placed on the top surface of the disk. The air vent sealing cover is rotated with respect to the disk either at a first position or a second position. At the first position, the hole of the air vent sealing cover is in correspondence to the air vent of the flow channel pattern to control the sample liquid delivery. At the second position, the air vent of the flow channel pattern is closed. The flow channel pattern includes at least one sample storage reservoir, at least one sample processing reservoir, and at least one communication channel which is in fluid communication between the sample storage reservoir and the sample processing reservoir. In alternative, the status of the hole of the air vent sealing cover is controlled by a control unit.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to a fluid sample separation device, and in particular to a disk-based fluid sample separation device that selectively allows a fluid sample contained in a sample storage reservoir to flow to a sample processing reservoir through the control of air vent and being subjected to a rotating motion. 
       BACKGROUND OF THE INVENTION 
       [0002]    Techniques for fluid sample separation are of wide applications, such as separation of cells, separation of fetal cells, cell separation for whole blood samples, and separation of endothelial colony forming cells (ECFC) contained in umbilical cord blood (UCB). 
         [0003]    For example, detection and quantification of cancer cells or rare cells present in body fluids are regarded as a potential indicator for clinical diagnoses, prognostication, and biomedicine research. For example, circulating tumor cells (CTC) are rare in the blood of patients with metastatic cancer, and it is possible to monitor the response of CTC to adjuvant therapy. Such rear cells must be first separated from the body fluids, before detection and quantification of these rare cells can be made. For such a purpose, various cell techniques have been developed. 
         [0004]    The cell separation techniques that are commonly used includes fluorescence activated cell separation (FACS), dielectrophoresis (DEP) cell separation, separation techniques that employ massively parallel microfabricated sieving devices, magnetically activated cell separation (MACS), and other techniques that uses optics and acoustics. Among these cell separation techniques, FACS and MACS are most often used. 
         [0005]    Although it is often used, FACS is disadvantageous in respect of high cost, difficulty in disinfection, and consuming a great amount of sample in the operation thereof. Contrary to FACS, MACS is efficient to obtain a major quantity of target cells in a short period with a reduced consumption of sample. However, these cells must be transferred to a slide or an observation platform before they can be observed with a microscope. Such a process of transfer often leads to a great loss of cells. 
         [0006]    Since MACS shows advantages in respect of high throughput, high performance, and simplified facility, it is often adopted in separation of fluid samples. Using immune cells to separate a desired component from a blood sample and the operation of immunofluorescence require multiple samples and manually-operated transfer, so that the result of detection is heavily dependent upon the skill of an operator, making it not fit for industrial use. 
       SUMMARY OF THE INVENTION 
       [0007]    In view of the above description of the conventional techniques, it is a major issue for this field to provide a fluid sample separation technique that realizes high throughput of cell selection, easy operation, low cost, simple facility, and excellent sensitivity and reliability. 
         [0008]    Thus, an objective of the present invention is to provide a disk based fluid sample separation device, which is of low cost, is easy for detection and observation, and has reduced cell loss, and is applicable to separate a labeled component from a fluid sample. 
         [0009]    Another objective of the present invention is to provide a disk based fluid sample separation device that is operated to selectively conduct a fluid sample contained in a sample storage reservoir to a sample processing reservoir by means of control realized by an air vent and rotary motion. 
         [0010]    A further objective of the present invention is to provide a disk based fluid sample separation device that is operated to separate, in a fluid sample, at least two types of cells, which are respectively labeled and not labeled with the immunomagnetic beads. 
         [0011]    The solution adopted in the present invention to achieve the above objectives is a microfluidic disk that forms therein a flow channel pattern. The flow channel pattern comprises at least one air vent. A sealing cover is set on a top surface of the microfluidic disk. The sealing cover forms at least one air passage. The sealing cover is rotatable with respect to the microfluidic disk between a first position, where the air passage of the sealing cover communicates the air vent of the flow channel pattern, and a second position, where the sealing cover closes the air vent of the flow channel pattern. The flow channel pattern comprises a sample storage reservoir, at least one sample processing reservoir, and a communication channel communicating between the sample storage reservoir and the sample processing reservoir. The sealing cover is operable through manual rotation or electrically-driven rotation to have the air passage of the sealing cover to align or close the air vent of a selected sample storage reservoir. In an embodiment of the present invention, the air vent of the sealing cover is replaced by a solenoid-controlled air vent structure. 
         [0012]    In a preferred embodiment of the present invention, at least one magnetic unit is set on a top of the sealing cover at a location corresponding to the sample processing reservoir of the microfluidic disk for providing a uniform magnetic force of predetermined magnitude on the sample processing reservoir. 
         [0013]    When the present invention is applied to separation of magnetically-labeled components contained in a fluid sample, it is capable of capturing all the magnetically-labeled components in whole blood cells. Further, the disk-based fluid sample separation device according to the present invention can be manufactured with a simple process, which can be carried out with laser machining, CNC machining, micromachining, or injection molding. Further, the material for manufacturing the disk is readily available, leading to an advantage of low manufacturing cost. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0014]    The present invention will be apparent to those skilled in the art by reading the following description of preferred embodiments thereof, with reference to the attached drawings, in which: 
           [0015]      FIG. 1  is a perspective view showing a disk-based fluid sample separation device constructed in accordance with a first embodiment of the present invention; 
           [0016]      FIG. 2  is an exploded view showing the disk-based fluid sample separation device of first embodiment of the present invention; 
           [0017]      FIG. 3  is a top plan view showing a microfluidic disk of the first embodiment of the present invention; 
           [0018]      FIG. 4  is a top plan view showing a sealing cover of the first embodiment of the present invention; 
           [0019]      FIG. 5  is a schematic view showing an air passage of the sealing cover of the present invention in alignment with an air vent of a sample storage reservoir to set the air vent in an open condition; 
           [0020]      FIG. 6  is a cross-sectional view showing the sealing cover of  FIG. 5  in a first position; 
           [0021]      FIG. 7  is a schematic view showing the sealing cover of the present invention being rotated by an angle to have the air passage aligning an air vent of another sample storage reservoir to set the air vent in an open condition; 
           [0022]      FIG. 8  is a cross-sectional view showing the sealing cover of  FIG. 7  in a second position; 
           [0023]      FIG. 9  is a schematic view showing the air vent of the sample storage reservoir of the present invention in a closed condition, whereby a fluid sample contained in the sample storage reservoir is not allowed to flow to a sample processing reservoir; 
           [0024]      FIG. 10  is a schematic view showing the air vent of the sample storage reservoir of the present invention in an open condition, whereby a fluid sample contained in the sample storage reservoir is acted upon by a centrifugal force to flow through a communication channel to the sample processing reservoir; 
           [0025]      FIG. 11  is a cross-sectional view taken along line  11 - 11  of  FIG. 1 ; 
           [0026]      FIG. 12-16  are schematic views demonstrating a fluid sample contained in the sample storage reservoir according to the present invention and secondary samples contained in secondary sample storage reservoirs conducted, under the control of air vents and subjected to rotating motion, to the sample processing reservoir; 
           [0027]      FIG. 17  is an exploded view showing a disk-based fluid sample separation device constructed in accordance with a second embodiment of the present invention; 
           [0028]      FIG. 18  is a top plan view showing a disk-based fluid sample separation device constructed in accordance with a third embodiment of the present invention; 
           [0029]      FIG. 19  is a cross-sectional view taken along line  19 - 19  of  FIG. 18 ; 
           [0030]      FIG. 20  is a cross-sectional view of an air passage opening/closing control unit of  FIG. 19  setting an air vent in an open condition; 
           [0031]      FIG. 21  is a cross-sectional view of the air passage opening/closing control unit of  FIG. 19  setting an air vent in a closed condition; 
           [0032]      FIG. 22  is a top plan view showing a disk-based fluid sample separation device constructed in accordance with a fourth embodiment of the present invention; and 
           [0033]      FIG. 23  is a cross-sectional view taken along line  23 - 23  of  FIG. 22 . 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0034]    With reference to the drawings and in particular to  FIG. 1 , which is a perspective view showing a disk-based fluid sample separation device constructed in accordance with a first embodiment of the present invention, and  FIG. 2 , which is an exploded view showing the disk-based fluid sample separation device of first embodiment of the present invention, the disk-based fluid sample separation device according to the present invention, generally designated at  100 , comprises a microfluidic disk  1 , which has a geometric center  11 , a top surface  12 , and a circumferential surface  13 , and is coupled, at the geometric center  11 , to a spindle of a rotation driving device  14 , whereby the microfluidic disk  1  is selectively driven by the rotation driving device  14  to rotate about the geometric center  11 , which serves as a rotation center, in a predetermined rotation direction I. 
         [0035]    The microfluidic disk  1  forms a flow channel pattern  2 . In the instant embodiment, the microfluidic disk  1  is composed of a bottom base board  15  and a flow channel pattern layer  16  formed on the bottom base board  15 . The flow channel pattern  2  is defined in and by the flow channel pattern layer  16 . The microfluidic disk  1  is covered by a sealing cover  3  set on the top surface  12  thereof. 
         [0036]    Referring also to  FIG. 3 , which is a top plan view of the microfluidic disk  1  shown in  FIG. 2 , the flow channel pattern  2  comprises at least one sample storage reservoir  21 , which is formed in the flow channel pattern layer  16  of the microfluidic disk  1  to store a fluid sample (such as a blood sample). The sample storage reservoir  21  is in fluid communication with at least one air vent  211 . The flow channel pattern  2  also comprises at least one secondary sample storage reservoir  21   a , which is formed in the flow channel pattern layer  16  of the microfluidic disk  1  for store a secondary sample (such as reaction reagent). Each of the secondary sample storage reservoirs  21   a  is set in fluid communication with a respective air vent  211   a.    
         [0037]    A plurality of secondary sample storage reservoirs  21   a  that comprises air vents  211   a  may be arranged on the microfluidic disk  1  as a circle centered at the geometric center  11 . Alternatively, secondary sample storage reservoirs comprising air vents may be arranged along inner and outer concentric circles on the microfluidic disk  1 . In the embodiment illustrated, a plurality of secondary sample storage reservoirs  21   a  that each comprises an air vent  211   a  is arranged as an outer circle in the flow channel pattern layer  16  of the microfluidic disk  1 , and a plurality of secondary sample storage reservoirs  21   b  that each comprises an air vent  211   b  is arranged as an inner, concentric circle in the flow channel pattern layer  16  of the microfluidic disk  1 . 
         [0038]    The flow channel pattern  2  further comprises at least one sample processing reservoir  22 . The sample processing reservoir  22  is located closer to the circumferential surface  13  of the microfluidic disk  1  than the sample storage reservoir  21  is. The sample processing reservoir  22  has a fluid inlet end  221  and a fluid outlet end  222 . The fluid inlet end  221  communicates through at least one communication channel  23 ,  23   a  with the sample storage reservoir  21  and the secondary sample storage reservoirs  21   a . The fluid outlet end  222  communicates with a capillary  24 . The capillary  24  has an opposite end extending to the circumferential surface  13  of the microfluidic disk  1  to form an opening  241 . 
         [0039]    In the instant embodiment, the bottom base board  15  and the flow channel pattern layer  16  are both made of acrylic resins, such as polymethylmethacrylate (PMMA), and the sealing cover  3  is made of a transparent material. Laser light, such as CO2 laser, is employed to machine the flow channel pattern layer  16  for forming the flow channel pattern  2 . The flow channel pattern layer  16  so formed may then be combined with the bottom base board  15 . Afterwards, the sealing cover  3  is set to cover the flow channel pattern layer  16  to thereby seal the top of the flow channel pattern  2 . 
         [0040]    Apparently, the flow channel pattern layer  16  can alternatively be formed as a multiple-layered structure by stacking or laminating multiple layers. Further, the microfluidic disk  1  can be alternatively made a single-layered structure and the material used is not limited to acrylic resins. The flow channel pattern  2  can alternatively be machined by for example other types of laser machining, or CNC machining, micromachining, and injection molding. 
         [0041]    The sealing cover  3  is positioned on the top surface of the microfluidic disk  1  and forms at least one air passage  31   a ,  31   b  (also see  FIGS. 2 and 4 ). The sealing cover  3  is rotatable with respect to the microfluidic disk  1 . For example, when the sealing cover  3  is rotated to a first angular position P 1  (also see  FIG. 5 , as well as the cross-sectional view of  FIG. 6 ), the air passage  31   a  of the sealing cover  3  is located exactly corresponding to the air vent  211   a  of the sample storage reservoir  21   a , thereby setting the air vent  211   a  in an open condition, while the air vents of the remaining sample storage reservoir are kept in a closed condition. Under this condition, the microfluidic disk  1  is driven to rotate about the geometric center  11 , and the air passage (such as  31   a ) of the sealing cover  3  is in alignment with the air vent (such as  211   a ) of a selected sample storage reservoir (such as  21   a ), the fluid sample stored in the selected sample storage reservoir  21   a  may be driven by a centrifugal force to flow through the communication channel  23   a  into the sample processing reservoir  22 . 
         [0042]    When the sealing cover  3  is rotated by a predetermined angle θ (also see  FIG. 7 , as well as the cross-sectional view of  FIG. 8 ), the air passage  31   b  of the sealing cover  3  is positioned to align the air vent  211   b  of the sample storage reservoir  21   b , thereby setting the air vent  211   b  in an open condition, while the air vents of the remaining sample storage reservoirs are kept closed. The number of the air passages formed in the sealing cover  3  may be varied as desired, and the locations where the air passages are formed are also variable as desired. Through the selective rotation of the sealing cover  3 , it is possible to selectively set the air vent of each individual sample storage reservoir in an open condition or a closed condition. 
         [0043]    Taking the sample storage reservoir  21  as an example, when the air vent  211  of the sample storage reservoir  21  is set in a closed condition (see  FIG. 9 ), the fluid sample W contained in the sample storage reservoir  21  is not allowed to flow to the sample processing reservoir  22 , whether the microfluidic disk  1  is kept standstill (not in rotation) or the microfluidic disk  1  is in rotation. On the other hand, when the air vent  211  of the sample storage reservoir  21  in an open condition (see  FIG. 10 ), if the microfluidic disk  1  is kept in standstill (not in rotation), the fluid sample W contained the sample storage reservoir  21  cannot flow to the sample processing reservoir  22 , but if the microfluidic disk  1  is driven and rotated, the fluid sample W contained in the sample storage reservoir  21  is acted upon by centrifugal force to flow into the sample processing reservoir  22 . 
         [0044]    With such an operation model, for an arrangement of a plurality of sample storage reservoirs, the angular displacement θ of the sealing cover  3  can be selected through rotation of the cover (see  FIG. 7 ) in order to selectively set the air vents of some of the sample storage reservoirs in a closed condition, while the air vents of the selected sample storage reservoirs are simultaneously opened to allow the fluid samples contained in the selected sample storage reservoirs to flow into the sample processing reservoir. Repeating the rotating and positioning process for the sealing cover  3  would allow the fluid sample contained in each of the sample storage reservoirs to be conducted into the sample processing reservoir (see  FIG. 10 ). 
         [0045]    Compared to a hydrophobic valve or a capillary valve adopted in the conventional centrifugal microfluidic platforms, the device of the present invention is less prone to influence by the nature of fluid sample, surface characteristics, size of communication channel, and rotational speed of microfluidic disk. 
         [0046]    Also referring to  FIG. 11 , which is a cross-sectional view taken along line  11 - 11  of  FIG. 1 , at least one magnetic unit  4  is additionally provided on the top of the sealing cover  3  at a location corresponding to the sample processing reservoir  22  of the microfluidic disk  1  for providing a predetermined magnetic field above the sample processing reservoir  22  of the microfluidic disk  1 . 
         [0047]    In an example application, the present invention is applied to separation of cells that are labeled with immunomagnetic beads. A fluid sample W with which the operation of cell separation is to be performed is first filled into the sample storage reservoir  21 . The fluid sample W contains two types of cell, one of which (target samples W 1 ) is labeled with immunomagnetic beads C. With the sealing cover  3  being angularly displaced to have the air passage  31   a  aligning the air vent  211  of the sample storage reservoir  21  and thus opening the air vent  211 , when the microfluidic disk  1  is driven by the rotation driving device  14  to rotate in a predetermined rotation direction I, the fluid sample W is acted upon by the centrifugal force induced by the rotation of the microfluidic disk  1  and thus flows from the sample storage reservoir  21  through the communication channel  23  into the sample processing reservoir  22 . Under this condition, the target samples W 1  that are labeled with immunomagnetic beads C contained in the fluid sample W are subjected to magnetic attraction induced by the magnetic unit  4  to collect at the underside of the sealing cover  3 . In the embodiment illustrated, the magnetic unit  3  comprises a rectangular array of magnets, which applies a uniform magnetic field of a predetermined intensity on the sample processing reservoir  22  of the microfluidic disk  1 . 
         [0048]    In another example of application, the present invention is used to separate for example MCF7 cells and Jurkat cells. It is apparent that the present invention is applicable to separation of fetal cells, separation of cells from whole blood sample, and separation of endothelial colony forming cells (ECFC) contained in umbilical cord blood (UCB). 
         [0049]      FIGS. 12-16  are schematic views demonstrating a fluid sample contained in the sample storage reservoir according to the present invention and secondary samples contained in secondary sample storage reservoirs conducted, under the control of air vents and subjected to rotating motion, to the sample processing reservoir. Firstly, the fluid sample is filled into the sample storage reservoir  21  and secondary samples are respectively filled into the respective secondary sample storage reservoirs  21   a ,  21   b  (see  FIG. 12 ). The sealing cover  3  is then rotated to have the air passage  31   b  of the sealing cover  3  aligning the air vent  211   a  of the sample storage reservoir  21   a . Afterwards, when the microfluidic disk  1  is put into rotation, the secondary sample contained in the secondary sample storage reservoir  21   a  is acted upon by a centrifugal force to flow through the communication channel  23   a  into the sample processing reservoir  22  (see  FIG. 13 ). 
         [0050]    After the secondary sample of the secondary sample storage reservoir  21   a  is completely received into the sample processing reservoir  22  (see  FIG. 14 ), the sealing cover  3  may be rotated again to have the air passage  31   a  of the sealing cover  3  aligning the air vent  211   b  of the sample storage reservoir  21   b  (see  FIG. 15 ). Under this condition, when the microfluidic disk  1  is put into rotation, the secondary sample contained in the secondary sample storage reservoir  21   b  is acted upon by a centrifugal force to flow through the communication channel  23   b  into the sample processing reservoir  22  (see  FIG. 16 ). As such, through sequential rotation of the sealing cover  3 , the fluid sample contained in the sample storage reservoir  22  and the secondary samples contained in the secondary sample storage reservoirs  21   a ,  21   b  can be individually conducted into the sample processing reservoir  22 . 
         [0051]      FIG. 17  is an exploded view showing a disk-based fluid sample separation device constructed in accordance with a second embodiment of the present invention, wherein the disk-based fluid sample separation device  100   a  of the second embodiment is formed of multiple layers stacked together, comprising a sealing cover  3 , three flow channel pattern layers  16   a ,  16   b ,  16   c , and a bottom base board  15 . 
         [0052]    In the previously discussed embodiments, the sealing cover  3  is positioned on the microfluidic disk  1  and is manually operable for rotation so as to have the air passage of the sealing cover  3  to correspond to or close an air vent of a selected sample storage reservoir. In another embodiment of the present invention, manual rotation of the sealing cover  3  is substituted by motor-driven rotation. Further, the air vent of the sealing cover  3  may be replaced by a solenoid controlled air vent structure. 
         [0053]    For example,  FIG. 18  is a top plan view showing a disk-based fluid sample separation device constructed in accordance with a third embodiment of the present invention, and  FIG. 19  is a cross-sectional view taken along line  19 - 19  of  FIG. 18 . In this embodiment, the disk-based fluid sample separation device, which is designated at  100   b , similarly comprises a microfluidic disk  5  that forms a flow channel pattern composed of a plurality of sample storage reservoirs  51  and/or secondary sample storage reservoir(s). A sealing cover  6  is set to cover the microfluidic disk  5 . The sealing cover  6  forms air vent channels  61  corresponding to the sample storage reservoirs  51  of the microfluidic disk  5 . Each air vent channel  61  has a top end to which an air passage opening/closing control unit  7  (such as a solenoid) is mounted and each air vent channel  61  has a bottom end  61   a  corresponding to and in fluid communication with the respective sample storage reservoir  51 . The top end of each the air vent channel  61  forms an air passage  61   b.    
         [0054]    Referring to  FIGS. 20 and 21 , which are cross-sectional views of the air passage opening/closing control unit  7  respectively showing the air vent in open and closed conditions as being controlled by the air passage opening/closing control unit, the air passage opening/closing control unit  7  comprises a solenoid  71 , an electromagnetic operation unit  72 , and a valve membrane  73 . When the solenoid  71  is excited by electrical power applied thereto, the electromagnetic operation unit  72  is operated to move the valve membrane  73  upwards, making the air vent channel  61  communicating an external air channel  61   c  (see  FIG. 20 ), whereby the fluid sample contained in the sample storage reservoir  51 , when acted upon by a centrifugal force, is allowed to flow out through the communication channel  51   a . When the solenoid  71  does not receive electrical power applied thereto, the electromagnetic operation unit  72  is not operated and the valve disk  73  returns to the original position to block the air vent channel  61  from the external air channel  61   c  (see  FIG. 21 ). Under this condition, the fluid sample stored in the sample storage reservoir  51  is prohibited from flowing out. 
         [0055]      FIG. 22  is a top plan view showing a disk-based fluid sample separation device  100   c  constructed in accordance with a fourth embodiment of the present invention, and  FIG. 23  is a cross-sectional view taken along line  23 - 23  of  FIG. 22 . In this embodiment, an arrangement that a single air passage opening/closing control unit  7  is operable for controlling multiple sample storage reservoirs  51  is provided. In other words, the sealing cover  6  has an air vent channel  61  that has a bottom end  61   a , which besides being in fluid communication with a sample storage reservoir  51 , is in communication with an extended air vent channel  2  for further communicating other sample storage reservoirs  51  through the extended air vent channel  62 , whereby when the solenoid  71  of the air passage opening/closing control unit  7  is excited by electrical power applied thereto, the fluid samples contained in the sample storage reservoirs  51  that are in communication with both the air vent channel  61  and the extended air vent channel  62  are allowed to flow out through the communication channel  51   a . When the solenoid  71  is not excited, the fluid sample contained in each of these sample storage reservoirs  51  is prohibited from flowing out. 
         [0056]    Although the present invention has been described with reference to the preferred embodiments thereof, it is apparent to those skilled in the art that a variety of modifications and changes may be made without departing from the scope of the present invention which is intended to be defined by the appended claims.