Abstract:
Provided are a blood-pretreating apparatus and method for removing corpuscles and abundant albumin from whole blood by use of microbeads. The blood-pretreating apparatus comprises a filter unit, packed with microbeads, for filtering out corpuscles and albumin through the microbeads from a blood sample introduced thereto; and a plasma storage unit for storing plasma free of corpuscles and albumin after the filtration in the filter unit

Description:
CROSS REFERENCE TO RELATED APPLICATION(S) 
       [0001]    This application claims the benefit of Korean Patent Application No. 10-2013-0078847, filed Jul. 5, 2013, which is hereby incorporated by reference herein in its entirety. 
       BACKGROUND OF THE INVENTION 
       [0002]    1. Technical Field 
         [0003]    The present invention relates to an apparatus and method for the pre-treatment of blood. More particularly, the present invention relates to a blood-pretreating apparatus and method for separating plasma from whole blood. 
         [0004]    2. Description of the Related Art 
         [0005]    Blood contains many proteins some of which are used as important biomarkers indicative of diseases or health conditions. In recent years, much development has been made of biochips in the form of strips designed to easily and quickly analyze biomarkers using small amounts of blood. 
         [0006]    Blood consists essentially of blood corpuscles and plasma. Corpuscles include erythrocytes, leucocytes, and platelets, accounting for more than about 40% of the volume of blood while plasma is comprised of water, proteins, lipids, carbohydrates, and minerals. 
         [0007]    A trace amount of proteins useful as biomarkers is present only in plasma. Accordingly, the application of plasma only, free of blood corpuscles, to biochips is advantageous in detecting protein biomarkers and can be done so with high sensitivity and high reproducibility. In this regard, various suggestions have been provided for separating plasma from blood on chips. For example, corpuscles are filtered through paper, glass fibers or microstructures on chips. In addition, corpuscle bias, separation and preparation are achieved using centrifugal force, electromagnetic force, and gravity. 
         [0008]    However, there is still been a demand for faster, simpler and more efficient techniques of removing corpuscles from a trace amount of non-diluted whole blood. 
         [0009]    Accounting for 50% or more of the amount of total plasma proteins, albumin may be highly apt to act as a potent noise in detecting biomarkers that are present in a trace amount. Hence, elimination of albumin contributes to the high sensitivity and reproducibility of biochips. 
         [0010]    Almost nowhere are means of removing albumin on biochips disclosed in previous literature. 
         [0011]    A related prior art is found in Korean Patent Application Publication No. 2013-0057720 (“Blood pre-treatment apparatus and Pretreatment method using the same”) describing that albumin is removed from whole blood in a short time to obtain plasma of low viscosity. 
         [0012]    Korean Patent Application Publication No. 2013-0057720 suggests forcible hemagglutination by which corpuscles can be quickly removed while plasma is obtained in a short time at improved efficiency. 
         [0013]    That is, Korean Patent Application Publication No. 2013-0057720 aims to obtain only plasma by inducing a blood sample to undergo hemagglutination after the removal of albumin 
       SUMMARY OF THE INVENTION 
       [0014]    Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide an apparatus and a method for the pre-treatment of blood in which corpuscles and abundant albumin are simultaneously removed from whole blood by use of microbeads. 
         [0015]    In order to achieve the above object, a blood-pretreating apparatus according to one preferred embodiment of the present invention comprises: a filter unit, packed with microbeads, for filtering out corpuscles and albumin through the microbeads from a blood sample introduced thereto; and a plasma storage unit for storing plasma free of corpuscles and albumin after the filtration in the filter unit 
         [0016]    Preferably, the filter unit may be configured to filter out corpuscles as the blood sample flows through microvoids formed between the packed microbeads. 
         [0017]    Preferably, the microbeads may be coated with a functional group designed to adsorb albumin to the microbeads, so that albumin is removed in the filter unit by being captured by the functional group of the packed microbeads. 
         [0018]    Preferably, the filter unit may have a cavity structure comprising a blood inlet and a plasma outlet wherein the blood inlet is positioned above the plasma outlet in view of gravitational direction and is greater in area than the plasma outlet. 
         [0019]    Preferably, the cavity structure may be of any one of a frusto-cone, a quadrangular frusto-pyramid, a stepped cylinder, and a triangular frusto-pyramid. 
         [0020]    Preferably, the side wall extending from the blood inlet to the plasma outlet may form an angle of 45 to 90 degrees with regard to the horizontal face. 
         [0021]    Preferably, the microbeads may be wetted in PBS (phosphate buffer solution) and then dried. 
         [0022]    Preferably, the microbeads may be pressurized by a load. 
         [0023]    Preferably, the microbeads may range in diameter from 50 μm to 200 μm. 
         [0024]    Preferably, the filter unit may be packed with the microbeads which are homogeneous or heterogeneous in diameter or which are a mixture of microbeads with homogeneous and heterogeneous diameters. 
         [0025]    Preferably, the microbeads may be any one of Reactive Blue2, Cibacron Blue 3G-A, and Cibacron Blue F3FA. 
         [0026]    Preferably, the filter unit may further comprise a cover for immobilizing the microbeads. 
         [0027]    Preferably, the filter unit and the plasma storage unit may be detachably fitted to each other. 
         [0028]    Preferably, the blood sample may flow as it is driven by capillarity and gravity. 
         [0029]    Preferably, the plasma storage unit may further comprise a biosensor for detecting a biomarker in the plasma. 
         [0030]    Preferably, the plasma storage unit may further comprise an air vent for helping the plasma to flow. 
         [0031]    Preferably, a hydrophilic surface treatment may be applied to the blood pre-treating apparatus. 
         [0032]    According to another preferred embodiment, the present invention provides blood-pretreating method, comprising: loading a blood sample to a filter unit packed with microbeads, said blood sample being deprived of corpuscles and albumin by the microbeads; and storing plasma in a plasma storage unit, said plasma being obtained by removing corpuscles and albumin from the blood sample. 
         [0033]    Having the above-illustrated structure, the apparatus of the present invention can remove corpuscles and albumin, simultaneously, from whole blood only by dripping a blood sample, and thus can be applied to a biochip for detecting a biomarker at high sensitivity and reproducibility. 
         [0034]    In addition, the apparatus and method according to the present invention can make a significant reduction in the blood amount required for analysis and in the time taken for plasma separation, and are applicable to various types of biochips, thus being qualified for general purpose uses. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0035]    The above and other objects, features and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: 
           [0036]      FIG. 1  is a cross sectional view illustrating the structure of a blood pre-treating apparatus according to one embodiment of the present invention; 
           [0037]      FIG. 2  is a perspective view of a blood pre-treating apparatus according to one embodiment of the present invention; 
           [0038]      FIG. 3  illustrates structures of the filter unit given in  FIGS. 1 and 2 ; 
           [0039]      FIG. 4  illustrates cover structures for immobilizing the microbeads given in  FIGS. 1 and 2 ; and 
           [0040]      FIG. 5  illustrates packing modes of the microbeads given in  FIGS. 1 and 2 . 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0041]    Below, a detailed description will be given of the blood-pretreating apparatus and method according to embodiments of the present invention with reference to the drawings. Prior to the detailed description, it should be noted that words and terms used in the specification and the claims must not be construed as only conventional or dictionary meanings. Thus, since the embodiments given in the specification and the structures shown in the drawings are only preferable embodiments, but cannot cover all the technical spirit of the present invention, it should be understood that there may be various equivalents and modifications alternative to the given embodiments at the time of the application of the present invention. 
         [0042]    With reference to  FIG. 1 , a cross sectional view is provided for illustrating the structure of a blood pre-treating apparatus  50  according to one embodiment of the present invention while  FIG. 2  is a perspective view of the blood pre-treating apparatus. 
         [0043]    As shown, the blood pre-treating apparatus  50  according to an embodiment of the present invention comprises a filter unit  10 , and a plasma storage unit  20 . 
         [0044]    The filter unit  10  is packed with predetermined microbeads  60  through which blood corpuscles and albumin are removed from a blood sample  1  (whole blood) introduced thereinto. Here, the filter unit  10  is formed on one side of a case body  40  while having a cavity structure comprising a blood inlet  10   a  to which whole blood  1  is introduced and a plasma outlet  10   b  from which plasma is released. 
         [0045]    As mentioned above, the microbeads  60  are packed within the filter unit  10 . They are coated with a functional group  70  designed to adsorb albumin to the surface of the microbeads  60 . Hence, albumin is captured by the functional group  70  of the microbeads  60  so that it can be removed in the filter unit  10 . 
         [0046]    After the removal of corpuscles and albumin by the filter unit  10 , the resulting plasma is stored in the plasma storage unit  20 . Herein, the plasma storage unit  20  may be established within the case body  40 . The one side of the plasma storage unit  20  is communicated with the plasma outlet  10   b.    
         [0047]    When the microbeads  60  are packed within the filter unit  10 , non-uniform microvoids are formed among the microbeads. The microvoids are significantly smaller in size than the microbeads  60 , and preferably designed to be as small as or smaller in size than corpuscles. To this end, the microbeads  60  preferably have a diameter of approximately 50 μm to 200 μm. 
         [0048]    In order to pack the microbeads  60  at high density within the filter unit  10 , a solution may be applied and then dried. For instance, a predetermined amount of dry microbeads  60  are added to the filter unit  10 , wetted with PBS (phosphate buffer solution) and then dried to obtain a high-density packing structure. Alternatively, pressurization with a load may be adopted to realize a high-density packing structure. For example, a predetermined amount of dry microbeads  60  are added to the filter unit  10 , and pressurized using a load which has a flat end. 
         [0049]    In another embodiment, the filter unit  10  may be structured to be detachably fitted to the plasma storage unit  20 . The detachable structures of the filter unit  10  and the plasma storage unit  20 , although not shown, will sufficiently be understood to those skilled in the art on the basis of the structure and description of  FIGS. 1 and 2 . For the detachable structure, the filter unit  10  and the plasma storage unit  20  may be made of different materials, and then may be integrated with each other. 
         [0050]    In the plasma storage unit  20 , a biosensor (not shown) for detecting a biomarker in plasma may be established. Although it is not graphically illustrated, the establishment of the biosensor will be understood to those having ordinary skill in the art. In addition, the plasma storage unit  20  may be provided with an air vent  30  for facilitating plasma flow within the plasma storage unit  20 . In  FIGS. 1 and 2 , the air vent  30  is positioned at a terminal region of the plasma storage unit  20  (that is, most distal to the plasma outlet  10   b ). However, the position may be changed as needed. 
         [0051]    The microbeads  60  packed within the filter unit  10  may be commercial beads configured for absorbing albumin For the microbeads  60 , for example, Reactive Blue2, Cibacron Blue 3G-A, Cibacron Blue F3FA, and a combination thereof may be used alone or in combination with various sub-synthetic compounds. However, the microbeads  60  are not limited to the above examples, and so long as it absorbs albumin, any microbead may be employed. The albumin within whole blood may be captured by the functional group  70  of the microbeads  60  within several seconds. A quantitative change of albumin can be monitored by, for example, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). 
         [0052]    Preferably, the flow of the whole blood  1  proceeds only by dropping. That is, the driving force of the blood flow within the apparatus may be preferably obtained from capillarity and gravity, without an external physical force. In order for blood to smoothly move without an external force, a hydrophilic surface treatment for facilitating a capillary flow may be additionally applied to the blood pre-treating apparatus  50 . For example, the apparatus may be surface oxidized by oxygen plasma technology, or may be coated with a surfactant or a protein. In addition or alternatively, the plasma storage unit  20  may be made of a hydrophilic material, such as glass, to facilitate capillary flow. 
         [0053]    When dripped to the blood inlet  10   a  in the blood pre-treating apparatus  50  according to an embodiment of the present invention, the whole blood  1  flows through the microvoids formed among the packed microbeads  60 , and comes out of the plasma outlet  10   b  to the plasma storage unit  20 . 
         [0054]    In this course, the whole blood is deprived of corpuscles by the microvoids, and albumin by the functional group of the microbeads, so that only plasma, free of corpuscles and albumin, is stored in the plasma storage unit  20 . 
         [0055]      FIG. 3  illustrates various structures, particularly, cavity structures of the filter unit  10  given in  FIGS. 1 and 2 . 
         [0056]    As shown in  FIG. 3 , the filter unit  10  may have various forms configured to allow the microbeads  60  to be packed therein, thereby serving to remove corpuscles and albumin simultaneously. 
         [0057]    In order to obtain an improvement both in the migration of the whole blood in the apparatus and in the corpuscle removal efficiency, it is preferred that the blood inlet  10   a  be positioned above the plasma outlet  10   b  in view of gravitational direction and that the area of the blood inlet  10  is larger than that of the plasma outlet  10   b.  In this structure, the blood movement proceeds faster as the cross area is gradually reduced in the direction of blood flow, and the corpuscles can be removed more efficiently by the bottleneck phenomenon thus set. 
         [0058]    As can be seen in  FIGS. 3   a,    3   b,    3   c  and  3   d,  the cavity of the filter unit  10  may be modified to be of a frusto-cone, a quadrangular frusto-pyramid, a stepped cylinder, or a triangular frusto-pyramid. In these cavity structures, the side wall may preferably form an angle (θ) of 45 to 90 degrees with regard to the horizontal face. 
         [0059]      FIG. 4  illustrates cover structures for immobilizing the microbeads given in  FIGS. 1 and 2 . 
         [0060]    With reference to  FIG. 4 , the filter unit  10  may be further provided with a cover  12  for immobilizing the microbeads  60 . When the blood pre-treating apparatus  50  is upside down after the microbeads  60  are packed within the cavity of the filter unit  10 , the microbeads do not maintain the packed condition, but escape towards the blood inlet  10   a.    
         [0061]    To prevent this, a cover  12  may be further established above the packed microbeads  60 . The cover  12  may be fixed to the filter unit  10 , as shown in  FIG. 4   a,  or may be set to be immobilized on the microbeads  60 . 
         [0062]      FIG. 5  illustrates packing modes of the microbeads given in  FIGS. 1 and 2 . 
         [0063]    Referring to  FIG. 5 , the microbeads  60  may be packed in various modes. The microbeads  60  may be homogeneous in diameter, as shown in  FIG. 5   a,  or completely heterogeneous in diameter, as shown in  FIG. 5   b,  or may be a mixture of microbeads with homogeneous and heterogeneous diameters. That is, only microbeads with a single diameter may be employed, or microbeads with different diameters are used in mixture. 
         [0064]    Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible without departing from the scope and spirit of the invention as disclosed in the accompanying claims. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 [Description of Reference Numerals] 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 10: Filter Unit 
                 12: Cover 
               
               
                   
                 20: Plasma Storage Unit 
                 30: Air vent 
               
               
                   
                 40: Case Body 
               
               
                   
                 50: Blood Pretreating Apparatus 
               
               
                   
                 60: Microbeads 
                 70: Reactor