Patent Publication Number: US-2011061472-A1

Title: Biochip system, method for determining sperm quality and method for separating sperm

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the priority benefit of U.S. provisional application Ser. No. 61/276,529, filed on Sep. 14, 2009. The entirety of the above-mentioned patent application is hereby incorporated by reference herein and made a part of specification. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a biochip system, and more particularly, to a lab-on-a-chip (LOC) for determining sperm quality or separating sperms. 
     2. Description of Related Art 
     In recent years, small-sized biochemical analysis systems have been vigorously developed and many microfluidics technologies have also been proposed for various applications. Because the small-sized analysis devices have the advantages of rapid analysis, low sample usage and space-saving, many analysis devices have been developed to be smaller and smaller, or even integrated into a single chip. Utilizing microfluidic chips to perform bio-medical inspection or analysis is also advantageous in reducing experimental errors arising from manual operation, increasing system stability, reducing power consumption and sample usage as well as saving labour force and time. 
     In general, the microfluidic chip is fabricated by using a semiconductor process to etch micro conduits in a glass or plastic substrate. An object to be inspected is allowed to flow in the micro conduits to sequentially perform the acts such as blend, separation and inspection. In other words, the entire function of the laboratory is integrated into the small sized cell to form a lab-on-a-chip (LOC). 
     SUMMARY OF THE INVENTION 
     Accordingly, the present invention is directed to a biochip system capable of evaluating the sperm motility and separating and collecting sperms with different motility by establishing flow fields with opposite directions in microfluidic regions. 
     The present invention is also directed to a method for determining sperm quality and separating sperms, in which the semen sample does not need to undergo any preprocessing. 
     In one aspect, the present invention provides a method for determining spew quality. At least one first microfluidic region and at least one second microfluidic region are provided. The first microfluidic region and the second microfluidic region meet at a junction. The second microfluidic region includes a shrunk portion. The width of the shrunk portion is sized to substantially allow only one sperm to pass therethrough, and a detector is disposed at the shrunk portion. A first flow field is formed in the first microfluidic region and a second flow field is formed in the second microfluidic region. The first flow field and the second flow field have different directions at the junction. A semen sample is loaded at a semen sample loading end. At least one sperm moves in the first microfluidic region against the direction of the first flow field. At least one sperm moves in the second microfluidic region along the direction of the second flow field. The detector generates a signal upon one sperm in the semen sample passing through the shrunk portion. 
     In another aspect, the present invention provides a method for separating sperms. At least one first microfluidic region and at least one second microfluidic region are provided. The first microfluidic region and the second microfluidic region meet at a junction. An end of the second microfluidic region is provided with a collecting portion. A first flow field is foamed in the first microfluidic region and a second flow field is formed in the second microfluidic region. The first flow field and the second flow field have different directions at the junction. A semen sample is loaded at a semen sample loading end. At least one sperm moves in the first microfluidic region against the direction of the first flow field. At least one sperm moves in the second microfluidic region along the direction of the second flow field so as to be collected by the collecting portion. In addition, the velocity of the first flow field in the first microfluidic region may be varied to collect sperms with different motility. 
     In still another aspect, the present invention provides a biochip system including at least one first microfluidic region, at least one second microfluidic region, and a detector. The first microfluidic region and the second microfluidic region meet at a junction. The first microfluidic region has a first flow field therein, and at least one sperm moves in the first microfluidic region against the direction of the first flow field. The second microfluidic region comprises a shrunk portion. The width of the shrunk portion is sized to substantially allow only one sperm to pass therethrough. The second microfluidic region has a second flow field therein, and, at the junction, the direction of the first flow field in the first microfluidic region is different from the direction of the second flow field in the second microfluidic region. At least one sperm moves in the second microfluidic region along the direction of the second flow field. The detector is disposed at the shrunk portion and is adapted to generate a signal upon one sperm passing through the shrunk portion. 
     In view of the foregoing, the biochip system of the present invention employs a particular flow field design to enable sperms in the semen sample to overcome the background velocity to move upstream, thereby facilitating detecting the number and concentration of motile sperms or separating sperms with specific motility. 
     Besides, in the method for determining sperm quality and separating sperms, a simple design is employed to generate desired flow fields, and the semen sample does not need to undergo any preprocessing such as dyeing process, marking process, or centrifuging process. Therefore, the biochip system of the present invention is capable of rapidly determining the sperm quality and evaluating the sperm motility in a simplified manner, and further separating and collecting sperms with different motility. 
     In order to make the aforementioned and other features and advantages of the present invention more comprehensible, embodiments accompanied with figures are described in detail below. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  is a perspective view of a biochip system according to a first embodiment of the present invention. 
         FIG. 1B  is a top, enlarged view of the area  110  of  FIG. 1A . 
         FIG. 2A  and  FIG. 2B  are schematic views showing a method for determining sperm quality carried out by the biochip system of the first embodiment. 
         FIG. 2B-1  is an enlarged view showing the path of the sperm passing through the shrunk portion of  FIG. 2B . 
         FIG. 2C  is a diagram showing the signal detected by the detector of one embodiment of the present invention. 
         FIG. 2C-1  is a partially enlarged view of the signal of  FIG. 2C . 
         FIG. 3  is a top view of microfluidic regions of a biochip system according to a second embodiment of the present invention. 
         FIG. 4  illustrates a microfluidic region design according to a third embodiment of the present invention. 
         FIG. 5A  and  FIG. 5B  illustrate the fourth embodiment of the biochip system that carries out the sperm quality determining method of the present invention. 
         FIG. 5C  illustrates the signal detected by a detector according to one embodiment of the present invention. 
         FIG. 6A  is a top view of microfluidic regions of a biochip system according to a fifth embodiment of the present invention. 
         FIG. 6B  is a top view of microfluidic regions of a biochip system according to a sixth embodiment of the present invention. 
         FIG. 6C  is a top view of microfluidic regions of a biochip system according to a seventh embodiment of the present invention. 
         FIG. 7A  is a top view of a biochip system according to an eighth embodiment of the present invention. 
         FIG. 7B  is a top, enlarged view of the junction  710  of  FIG. 7A . 
         FIG. 7C  is a top view of a biochip system according to a ninth embodiment of the present invention. 
         FIG. 8  is a comparison diagram of the percentage of motile sperm in a semen sample prior to and after a separation process using the biochip system  700  of  FIG. 7A  and  FIG. 7B . 
     
    
    
     DESCRIPTION OF THE EMBODIMENTS 
     The present invention provides a biochip system having microfluidic regions, which at least includes a substrate having an upper surface and microfluidic regions formed on the upper surface of the substrate. The biochip system employs microfluidics technology such that sperms can move upstream to a detecting region where a detector is disposed to cause the detector to generate an electrical signal. In this way, the detector detects the number of sperms that move upstream a fixed distance within a fixed time period, which reflects the number and concentration of motile sperms. In the biochip system described below, the microfluidic regions are implemented as micro conduits formed in a material layer over the substrate. It is noted that this is for the purposes of illustration only and should not be regarded as limiting. The microfluidic regions of the present invention could be fabricated in any manner as would be appreciated by those skilled in the art and therefore should not be limited to the particular embodiments described below. 
       FIG. 1A  is a perspective view of a biochip system according to a first embodiment of the present invention.  FIG. 1B  is a top, enlarged view of the area  110  of  FIG. 1A . 
     Referring to  FIG. 1A , the biochip system  100  includes a substrate  102  and a material layer  104  disposed over an upper surface  102   a  of the substrate  102 . At least one microfluidic region  112 , at least one microfluidic region  114 , and at least one microfluidic region  116  are formed on the upper surface  102   a  of the substrate  102 . Only one microfluidic region  112 , one microfluidic region  114  and one microfluidic region  116  are illustrated in the drawings. It is noted that this is for the purposes of description only and the number and shape of the microfluidic regions described herein should not be regarded as limiting. The microfluidic regions  112 ,  114 ,  116  are, for example, formed in the material layer  104  and the substrate  102  serves as a bottom of the microfluidic regions  112 ,  114  and  116 . The material of the substrate  102  is, for example, glass. The material of the material layer  104  may be a transparent bio-compatible material, for example, a soft transparent polymer material such as polydimethylsiloxane (PDMS). Because the soft and transparent PDMS material can easily be adhered onto the glass substrate and is resilient, a liquid can be directly injected into the material layer  104  without leakage. As such, with the microfluidic regions  112 ,  114  and  116  made of PDMS, the liquid in the material layer  104  is allowed to be observed and trapped at the same time. 
     In addition to the material layer  104 , other components, such as reservoirs  106   a ,  106   b  and  106   c , can be formed on the substrate  102 . The reservoirs  106   a ,  106   b  and  106   c  are, for example, disposed on a surface of the material layer  104  in communication with the microfluidic regions  112 ,  114  and  116 , respectively. The reservoirs  106   a ,  106   b  and  106   c  can be used to store or collect samples, reagents or buffer solutions. 
     In the area  110  shown in  FIG. 1B , the microfluidic regions  112 ,  114  and  116  meet at a junction  118  such that they form microfluidic conduits that have a T-shaped configuration and communicate with one another. The microfluidic region  112  and the microfluidic region  114  extend, for example, in the same direction and are connected to the junction  118 , while the microfluidic region  116  is connected to the junction  118 , for example, at an angle with respect to the microfluidic regions  112  and  114 . 
     The microfluidic region  112  has an end  112   a  positioned at a side opposite to the junction  118 . The end  112   a  acts, for example, as a semen sample loading end and communicates with the reservoir  106   a . The reservoir  106   a  contains, for example, a semen sample that does not undergo any preprocessing. The length L 1  of the microfluidic region  112  is about within the range from 0.05 mm to 40 mm, and the width W 1  of the microfluidic region  112  is about within the range from 5 um to 10000 um. The microfluidic region  114  has an end  114   a  positioned at a side opposite to the junction  118 . The end  114   a  acts, for example, as an exit end for moving sperms and communicates with the reservoir  106   b . The reservoir  106   b  contains, for example, RPMI  1640  nutrient solution. The length L 2  of the microfluidic region  114  is about within the range from 0.01 mm to 40 mm, and the width W 2  of the microfluidic region  114  is about within the range from 5 um to 10000 um. The microfluidic region  116  has an end  116   a  positioned at a side opposite to the junction  118 . The end  116   a  acts, for example, as a flow field source end to provide a buffer solution and communicates with the reservoir  106   c . The reservoir  106   c  contains, for example, the buffer solution that is prepared by mixing the RPMI  1640  nutrient solution and seminal plasma, wherein the seminal plasma may be used to prevent the sperm from adhering to the conduits. The length L 3  of the microfluidic region  116  is about within the range from 0.1 mm to 40 mm, and the width W 3  of the microfluidic region  116  is about within the range from 5 um to 10000 um. In addition, the conduit depth of the microfluidic regions  112 ,  114  and  116  in the material layer  104  is about within the range from 5 um to 100 um. 
     The microfluidic region  114  includes a shrunk portion  120  positioned, for example, adjacent the joining area between the microfluidic region  116  and the microfluidic region  114 . In one embodiment, the shrunk portion  120  may be a channel extending in parallel with the extending direction of the microfluidic region  114  and the extending channel of the shrunk portion  120  has a length L A . The shrunk portion  120 , acting as a detecting region, has a smaller conduit width W A  such that only one sperm is allowed to pass therethrough at one time. That is, a part of the conduit wall of the microfluidic region  114  adjacent the junction  118  is recessed inwardly to narrow the conduit width at this part. Since the size of the sperm cell is about 2 um to 4 um, the conduit width W A  of the shrunk portion  120  can be designed to be about within 5 um to 20 um. A detector (not shown) is, for example, disposed at the shrunk portion  120  for detecting the single sperm passing through the shrunk portion  120  each time. The detector may be a counter designed under the Coulter principle to calculate the total number of sperms passing through the shrunk portion  120 . 
     In one embodiment, at the junction  118 , the microfluidic region  116  can be connected to the microfluidic regions  112  and  114  in a direction perpendicular or not perpendicular to the extending direction of the microfluidic regions  112  and  114 . As shown in  FIG. 1B , the conduit walls of the microfluidic region  116  and the microfluidic region  112  form, for example, an angle θ 1  at a joining area therebetween. The conduit walls of the microfluidic region  116  and the microfluidic region  114  around the shrunk portion  120  form, for example, an angle θ 2  at a joining area therebetween. The angle θ 1  and the angle θ 2  may be arbitrary values and may be equal or different. It is not intended to limit the angles to any particular value in the present invention. 
     It is noted that the microfluidic regions  112 ,  114  and  116  can have stable flow fields  122 ,  124  and  126 , respectively, by controlling the velocity of the fluid in the biochip system  100  of the first embodiment. Specifically, the buffer solution is injected via the end  116   a  into the microfluidic region  116  as a flow field source to provide a flow field  126  with high flow velocity in the microfluidic region  116 . When flowing from the end  116   a  to the junction  118 , the buffer solution is separated into two parts, one of which flows from the junction  118  to the end  112   a  to form a flow field  122 , and the other of which flows from the junction  118 , through the shrunk portion  120  and to the end  114   a  to form a flow field  124 . That is, the direction of the flow field  122  is opposite to the direction of the flow field  124 . 
     The velocity of the flow field  122  in the microfluidic region  112  is considered as a background flow velocity, which is, for example, a threshold for determining or screening motility of sperm in a semen sample. In one embodiment, when the semen sample is loaded at the end  112   a  of the microfluidic region  112 , sperms in the semen sample move in a direction against the flow field  122  in the microfluidic region  112 . When the moving sperms can overcome the velocity of the flow field  122 , the sperms can move upstream in the microfluidic region  112  toward the junction  118 . After passing through the junction  118 , the sperms are carried by the flow field  124  in the microfluidic region  114  toward a second end and to pass through the detecting region at the shrunk portion  120  in the direction of the flow field  124 . On the contrary, when the moving sperms cannot overcome the velocity of the flow field  122 , the sperms are flushed downstream with the buffer solution in the microfluidic region  112 . In other words, those sperms with a certain level of motility can be detected or screened out by setting a proper velocity of the flow field  122  such that the motile sperms can overcome the flow field  122  to move upstream toward the junction  118  and can be detected or screened out by the detector disposed at the shrunk portion  120 . In general, the moving speed of sperms is about within the range from 1 um/s to 70 um/s. The maximum velocity of the flow field  122  is substantially less than the maximum moving speed of the sperms. For example, the velocity of the flow field  122  can be set to be within the range from 5 um/s to 80 um/s. 
     In addition, the velocity of the flow field  126  in the microfluidic region  116  is substantially greater than the moving speed of the sperms to prevent the sperms passing through the junction  118  from entering the microfluidic region  116 . The maximum velocity of the flow field  126  is, for example, about within the range from 80 um/s to 150 um/s. The buffer solution flowing from the microfluidic region  116  into the microfluidic region  114  can generate a flow field  124  with high velocity at the time of passing through the shrunk portion  120 . The velocity of the flow field  124  is, for example, greater than the velocity of the flow field  122  and greater than the moving speed of the sperms, such that the sperms moving upstream to the junction  118  can be carried to pass through the shrunk portion  120  rapidly. The maximum velocity of the flow field  124  is, for example, about within the range from 80 um/s to 150 um/s. In one embodiment, the maximum velocity of the flow field  124  is 100 um/s. 
     The velocity of the flow field  122 ,  124  and  126  can be adjusted by changing the height of liquid in the reservoirs  106   a ,  106   b  and  106   c  to generate different hydrostatic pressure or by modifying the width of the microfluidic regions  112 ,  114  and  116 . In one embodiment, the height of liquid in the reservoir  106   c  is greater than the height of liquid in the reservoir  106   b  and, therefore, the buffer solution in the reservoir  106   c  can flow from the microfluidic region  116  into the microfluidic regions  112  and  114 , thereby establishing the flow field with the desired direction. 
     The method for determining sperm quality will now be described below in conjunction with the biochip system  100  illustrated in  FIG. 1A  and  FIG. 1B . However, embodiments described below are for the purposes of illustration only and should not be regarded as limiting. 
       FIG. 2A  and  FIG. 2B  are schematic views showing a method for determining sperm quality carried out by the biochip system of the first embodiment.  FIG. 2C  is a diagram showing the signal detected by the detector of one embodiment of the present invention.  FIG. 2B-1  is an enlarged view showing the path of the sperm passing through the shrunk portion of  FIG. 2B .  FIG. 2C-1  is a partially enlarged view of the signal of  FIG. 2C . 
     The detector  200  used in  FIG. 2A  and  FIG. 2B  is, for example, a counter designed under the Coulter principle, which includes an electrode  202  and an electrode  204  disposed in the microfluidic region  114  and the microfluidic region  116 , respectively. The detector  200  provides a constant current to measure an impedance variation caused by sperms  210  passing through the shrunk portion  120  within a fixed time period. The measuring results are shown in  FIG. 2C . 
     Referring to  FIG. 2A  and  FIG. 2C , at an initial state (t= 0 ), an initial sample containing sperms  210  is loaded at the end  112   a  of the microfluidic region  112 . At least one of the sperms  210  is able to overcome the flow field  122  of the microfluidic region  112  to move upstream toward the junction  118 . Once passing through the microfluidic region  112  and reaching the junction  118  after a period of time, the sperm  210  is carried by the flow field  124  toward the end  114   a  and to pass through the shrunk portion  120 , causing the detector  200  to generate an electrical signal. It is noted that, due to the narrow width of the conduit at the shrunk portion  120 , when the sperm  210  passes through the micro shrunk portion  120 , it causes an increase of resistance. Under the condition that the two sides of the shrunk portion  120  are provided with a constant current, the detector  200  can generate a voltage pulse  121  as a detecting signal while the sperm  210  passes through the shrunk portion  120 . 
     After measuring for a specific time period (t=t 0 ), as shown in  FIG. 2B  and  FIG. 2C , each sperm  210  passing through the shrunk portion  120  causes a pulse in the signal detected by the detector. As such, the sperm number and concentration can be calculated by detecting the electrical signal within the specific time period. In other words, each time only one sperm  210  is able to pass through the extending channel of the shrunk portion  120  and, therefore, the voltage signal detected by the detector can have only one pulse  212  during the relatively short time period when the sperm  210  is being passing through the shrunk portion  120 . The continuous multiple pulses  212  as shown in  FIG. 2C  indicate that multiple sperms  210  pass through the extending channel of the shrunk portion  120  one by one. Therefore, each sperm  210  passing through the shrunk portion  120  causes a corresponding pulse signal  212  detected by the detector over time. 
     As shown in  FIG. 2B-1  and  FIG. 2C-1 , it is noted that, because the shrunk portion  120  has the extending channel with length L A , the electrical signal detected when a single sperm  210  passes through the shrunk portion  120  can provide information relating to the sperm speed, size and vibration according to a movement path along which the same sperm moves in the shrunk portion  120  or a manner in which the sperm vibrates. In other words, when a single sperm passes through the shrunk portion  120 , different sperms have different motility and flagellum vibration manners and therefore have different movement paths. As such, when one single sperm passes through the shrunk portion  120 , the pulse signal detected is different, thereby providing characteristics of the corresponding sperm passing through the shrunk portion  120 . 
     For example, as shown in  FIG. 2C-1 , each voltage pulse  212  generated by the detector  200  at the time the sperm  210  passes through the shrunk portion  120  is a pulse signal that maintains a high voltage for a period of time rather than having only one peak. The duration  212   a  of the pulse  212  depends on, for example, the moving speed of the sperm  210  passing through the shrunk portion  120 . In addition, each voltage pulse  212  has multiple fluctuations  212   b  on its wave crest. The fluctuations  212   b  of the pulse  212  depend on, for example, the manner in which the sperm  210  vibrates in the shrunk portion  120 . The amplitude of the pulse  212  depends on, for example, the size of the sperm  210  passing through the shrunk portion  120 . 
       FIG. 3  is a top view of microfluidic regions of a biochip system according to a second embodiment of the present invention. 
     In another embodiment, the biochip system can further include a collecting portion  302  in communication with the microfluidic region  114 . The collecting portion  302  is, for example, connected to the end  114   a  of the microfluidic region  114 , for collecting the sperms that have a sperm motility sufficient to overcome the flow field  122  in the microfluidic region  112  to pass through the junction  118  and shrunk portion  120  and are carried to the end  114   a . The collecting portion  302  may also be the reservoir  106   b  of  FIG. 1A  for storing the sperms that move upstream through the junction  118  and are carried to the end  114   a  by the flow field  124 . Therefore, in addition to being able to detect the sperm number and sperm concentration in the manner illustrated in  FIG. 2A  and  FIG. 2B , the biochip system of the present embodiment can also separate the motile sperms from the initial sample. Besides, the biochip system can further be provided with an observation device  304  at the end  114   a , for example, a microscope and a charge coupled device (CCD) for observing the morphology of the collected sperms. Because the collected sperms are able to overcome the background flow field  122  in the microfluidic region  112  to move upstream, the sperm motility can also be evaluated by setting the velocity of the background flow field  122 . 
     While the biochip system is illustrated as forming three microfluidic regions with different flow velocity on the upper surface of the substrate in the above embodiments, it is noted that this is for the purposes of illustration only and should not be regarded as limiting. Rather, in other embodiments, the microfluidic region can be configured differently, as described below. 
       FIG. 4  illustrates a microfluidic region design according to a third embodiment of the present invention. 
     As shown in  FIG. 4 , in one embodiment, two microfluidic regions  402  and  404  in communication with each other are formed on the upper surface of the substrate. For example, the microfluidic region  402  and the microfluidic region  404  extend in the same direction and are connected to a junction  408 . Another microfluidic region  406  is connected to the junction  408  and the microfluidic region  406  is not located on the plane on which the microfluidic regions  402  and  404  are located. The microfluidic region  406  is, for example, a component that can provide a high velocity flow field. The microfluidic region  406  may be an injector that injects the buffer solution into the microfluidic regions  402  and  404  from above the microfluidic regions  402  and  404 . 
     Similarly, when the externally injected buffer solution flows from the microfluidic region  406  to the junction  408 , it forms a high velocity flow field  426  and is separated into two parts at the junction  408 . One part of the buffer solution flows from the junction  408  toward the microfluidic region  402  to form a flow field  422 , and the other part of the buffer solution flows from the junction  408 , through the shrunk portion  410 , toward the microfluidic region  404  to form a flow field  424 , thus resulting in the two flow fields  422  and  424  with opposite directions. As such, when a sperm loaded at the end  402   a  of the microfluidic region  402  is able to overcome the flow field  422  of the microfluidic region  402  to move upstream toward the junction  408 , the sperm can be carried by the flow field  424  toward the microfluidic region  404  and to pass through the detecting region at the shrunk portion  410 , causing the detector to generate an electrical signal. 
     The shrunk portion is described as having an extending channel with a length L A  in the above embodiments. However, this is for the purposes of illustration only and should not be regarded as limiting. It would be understood by those skilled in the art that the shrunk portion may also be a structure without an extending channel as long as the conduit width at the shrunk portion is sized to allow only one sperm to pass therethrough at one time so that the shrunk portion can be used as a detecting region. Another structure of the shrunk portion is described below with reference to a fourth embodiment of the biochip system. It should be understood that the shrunk portion of the biochip system of the fourth embodiment can also be applied in any one of the other embodiments and therefore should not be limited to this particular application as illustrated in the drawings. 
       FIG. 5A  and  FIG. 5B  illustrate the fourth embodiment of the biochip system that carries out the sperm quality determining method of the present invention.  FIG. 5C  illustrates the signal detected by a detector according to one embodiment of the present invention. It is noted that, in  FIG. 5A  and  FIG. 5B , elements that are the same as in FIG.  2 A and  FIG. 2B  are referenced by the same numerals and explanation thereof is not repeated herein. 
     In the fourth embodiment, the main elements of the biochip system of  FIG. 5A  and  FIG. 5B  are substantially the same as that in  FIG. 2A  and  FIG. 2B . The main difference lies in the configuration of the shrunk portion  520 . As shown in  FIG. 5A , the shrunk portion  520  is an extending channel without a specific length. In other words, the shrunk portion  520  is, for example, a structure with an aperture, which likewise allows only one single sperm to pass therethrough at one time. The conduit wall at the interconnecting area between the microfluidic region  114  and the microfluidic region  116  is recessed inwardly at a position adjacent the junction  118  to form a cusp, thus resulting in a narrow conduit width at the shrunk portion  520 . 
     Similarly, the detector  200  used in  FIG. 5A  and  FIG. 5B  is, for example, a counter designed under the Coulter principle, which includes an electrode  202  and an electrode  204  disposed in the microfluidic region  114  and the microfluidic region  116 , respectively. The detector  200  provides a constant current to measure an impedance variation caused by sperms  510  passing through the shrunk portion  520  within a fixed time period. The measuring results are shown in  FIG. 5C . 
     Referring to  FIG. 5A  and  FIG. 5C , at an initial state (t= 0 ), an initial sample containing sperms  510  is loaded at the end  112   a  of the microfluidic region  112 . At least one of the sperms  510  is able to overcome the flow field  122  of the microfluidic region  112  to move upstream toward the junction  118 . The sperm  210  is then carried by the flow field  124  toward the end  114   a  and to pass through the shrunk portion  520 . At the moment when the sperm  510  passes through the shrunk portion  520 , the sperm  510  causes an increase of resistance. Therefore, the detector  200  generates a voltage pulse  512  as a detecting signal while the sperm  510  passes through the shrunk portion  520 . 
     After measuring for a specific time period (t=t 0 ), as shown in  FIG. 2B  and  FIG. 2C , each sperm  510  passing through the shrunk portion  520  causes a pulse  512  in the signal detected by the detector. As such, the sperm number and concentration can be calculated by detecting the electrical signal within the specific time period. 
     It is to be understood that the present invention can be implemented in other embodiments other than the embodiments described above. In the above embodiments, the two flow fields at the junction have opposite directions, and the microfluidic region connected to the semen sample loading end and the microfluidic region connected to the motile sperm exit end are arranged and connected along a same straight line. However, this is for the purposes of illustration only and should not be regarded as limiting. In other embodiments, the microfluidic region connected to the semen sample loading end and the microfluidic region connected to the motile sperm exit end can be arranged and connected in any suitable fashion, as long as at least two flow fields with opposite directions are formed at the junction, which are described below by way of examples. 
       FIG. 6A  is a top view of microfluidic regions of a biochip system according to a fifth embodiment of the present invention.  FIG. 6B  is a top view of microfluidic regions of a biochip system according to a sixth embodiment of the present invention.  FIG. 6C  is a top view of microfluidic regions of a biochip system according to a seventh embodiment of the present invention. For clarity,  FIG. 6A ,  FIG. 6B  and  FIG. 6C  mainly show the configurations of the microfluidic region connected to the semen sample loading end and the microfluidic region connected to the motile sperm exit end, without showing the microfluidic region that acts as the flow field source. Besides, like elements are referenced by like numerals and explanation thereof is therefore not repeated herein. 
     Referring to  FIG. 6A , in the fifth embodiment, a microfluidic region  602  is connected to a microfluidic region  604  at a junction  608 . The microfluidic region  602  has an end  602   a  at a side opposite to the junction  608 . The end  602  is, for example, used as a semen sample loading end. The microfluidic region  604  has an end  604   a  at a side opposite to the junction  608 . The end  604   a  is, for example, used as an exit end for motile sperms. The microfluidic region  604  includes a shrunk portion  610  which is, for example, positioned adjacent the junction. Besides, the biochip system of the fifth embodiment further includes another microfluidic region (not shown) connected to the junction  608 , acting as a flow field source. 
     The microfluidic region  602  and the microfluidic region  604  are interconnected to form a U-like configuration. The microfluidic region  602  and the microfluidic region  604  are, for example, arranged in parallel except for the areas adjacent the junction  608 . Namely, the part of microfluidic region  602  adjacent the end  602   a  and the part of microfluidic region  604  adjacent the end  604   a  extend in the same direction. By controlling the flow velocity, stable flow fields  612  and  614  are formed in the microfluidic regions  602  and  604 , respectively. The buffer solution injected from the flow field source end flows from the junction  608  to the microfluidic region  602  and the microfluidic region  604 , respectively, and, therefore, the flow field  612  and the flow field  614  have different directions at the junction  608 . As such, motile sperms in the semen sample are able to move against the flow field  612  to pass through the junction  608 , and are then carried by the flow field  614  in the microfluidic region  604  toward the end  604  and to pass through the detecting region at the shrunk portion  610 . 
     Referring to  FIG. 6B , the main elements of the biochip system of the sixth embodiment are similar to that of the fifth embodiment. The main difference lies in the angle θ 3  between the microfluidic region  602  and the microfluidic region  604 . The microfluidic region  602  and the microfluidic region  604  may also be arranged in a nonparallel fashion thus forming an angle θ 3  at the junction  608 . The angle θ 3  may be of any suitable values. 
     In addition, the present invention is not intended to limit the number of the microfluidic regions to any particular number described herein. Referring to  FIG. 6C , the main elements of the biochip system of the seventh embodiment of the present invention are similar to that of the fifth embodiment. The main difference lies in the number of the microfluidic regions connected to the semen sample loading end. In the seventh embodiment, the microfluidic regions  602 ,  604  and  620  are connected at the junction  608 . The microfluidic region  620  has an end  620  acting as a semen sample loading end. The buffer solution injected from the flow field source end flows from the junction  608  to the microfluidic regions  602 ,  620  and  604 , respectively. Therefore, a stable flow field  622  is also formed in the microfluidic region  620 , and the flow fields  612 ,  622  and  614  have different directions at the junction  608 . In other words, motile sperms in the semen sample are able to move against the flow field  612  in the microfluidic region  602  or move against the flow field  622  in the microfluidic region  620  toward the junction  608 . In one embodiment, the end  602   a  and end  620   a  both used as the semen sample loading end may or may not be connected to each other. 
     While the biochip system is illustrated as having two microfluidic regions connected to the semen sample loading end in  FIG. 6C , it is to be understood that the biochip system can have multiple microfluidic regions connected to the motile sperm exit end or multiple microfluidic regions connected to the flow field source end in other embodiments. As would be appreciated by those skilled in the art upon reading the foregoing description, various elements of the biochip system can be modified or used in combination without departing from the spirit and scope of the present invention, which are therefore not described herein further. 
     The present invention further provides a biochip system with microfluidic regions, which at least includes a substrate with an upper surface and a plurality of microfluidic regions formed on the upper surface of the substrate. This biochip system employs the microfluidics technology to design the flow field such that sperms can move upstream a fixed distance before being carried to a collecting end and the sperms with different motility can be screened out or separated by controlling the velocity of a background flow field. 
       FIG. 7A  is a top view of a biochip system according to an eighth embodiment of the present invention.  FIG. 7B  is a top, enlarged view of the junction  710  of  FIG. 7A . 
     Referring to  FIG. 7A , the biochip system  700  at least includes a substrate and a material layer  704  disposed over an upper surface of the substrate  702 . Microfluidic regions  712 ,  714  and  716  are formed on the upper surface of the substrate  702 . The microfluidic regions  712 ,  714  and  716  are, for example, fowled in the material layer  704  and the substrate  702  serves as a bottom of the microfluidic regions  712 ,  714  and  716 . The material of the substrate  702  is, for example, glass. The material of the material layer  704  may be a transparent bio-compatible material, for example, a soft transparent polymer material such as polydimethylsiloxane (PDMS). In one embodiment, the biochip system  700  may further include reservoirs  706   a ,  706   b  and  706   c  for storing samples, reagents or buffer solutions. The reservoirs  706   a ,  706   b  and  706   c  are, for example, disposed on a surface of the material layer  704  and communicate with the microfluidic regions  712 ,  714  and  716 , respectively. 
     Referring to  FIG. 7A  and  FIG. 7B , the microfluidic regions  712 ,  714  and  716  are fluidly connected with one another to form a T-shaped microfluidic conduit. The microfluidic region  712  and the microfluidic region  714  extend, for example, in the same direction and are connected to each other, while the microfluidic region  716  is connected to the junction  710 , for example, at an angle perpendicular to the microfluidic regions  112  and  114 . 
     The microfluidic region  712  has an end  712   a  acting, for example, as a semen sample loading end and communicating with the reservoir  706   a . The reservoir  706   a  contains, for example, a semen sample that does not undergo any preprocessing. The length L 4  of the microfluidic region  712  is about within the range from 0.05 mm to 40 mm, and the width W 4  of the microfluidic region  712  is about within the range from 5 um to 10000 um. The microfluidic region  714  has an end  714   a  acting, for example, as an exit end for moving sperms and communicating with the reservoir  706   b . The reservoir  706   b  contains, for example, RPMI  1640  nutrient solution. The length L 5  of the microfluidic region  714  is about within the range from 0.01 mm to 40 mm, and the width W 5  of the microfluidic region  714  is about within the range from 10 um to 10000 um. The microfluidic region  716  has an end  716   a  acting, for example, as a flow field source end to provide a buffer solution and communicating with the reservoir  706   c . The reservoir  706   c  contains, for example, the buffer solution that is prepared by mixing the RPMI  1640  nutrient solution and seminal plasma, where the seminal plasma may be used to prevent the sperm from adhering to the conduits. The length L 6  of the microfluidic region  716  is about within the range from 0.01 mm to 40 mm, and the width W 6  of the microfluidic region  716  is about within the range from 5 um to 10000 um. In addition, the conduit depth of the microfluidic regions  712 ,  714  and  716  in the material layer  704  is about within the range from 5 um to 1000 um. 
     As shown in  FIG. 7B , in the biochip system  700 , the buffer solution injected via the end  716   a  provides a flow field  726  with high flow velocity in the microfluidic region  716 . When flowing from the end  716   a  to the junction  718  of the microfluidic regions  712 ,  714  and  716 , one part of the buffer solution flows to the end  712   a  to form a flow field  722 , and the other part of the buffer solution flows to the end  714   a  to form a flow field  724 . The direction of the flow field  722  is opposite to the direction of the flow field  724 . In one embodiment, when the semen sample is loaded at the end  712   a  of the microfluidic region  712 , sperms in the semen sample must overcome the background flow velocity of the flow field  722  before moving upstream toward the junction  710 . Once passing through the junction  710 , the sperms can pass through the microfluidic region  714  and reach the collecting end rapidly with the aid of the high velocity flow field  724 . 
     The maximum velocity of the flow field  722  is substantially less than the maximum moving speed of the sperms, and, for example, can be set to be about within the range from 5 um/s to 80 um/s. The maximum velocity of the flow field  724  is greater than the moving speed of the sperms, and, for example, is about within the range from 80 um/s to 150 um/s. In one embodiment, the maximum velocity of the flow field  724  is 100 um/s. The maximum velocity of the flow field  726  is, for example, about within the range from 80 um/s to 150 um/s. 
     In one embodiment, sperms with different motility can be separated by setting different velocity of the flow field  722 . For example, when the maximum velocity of the flow field  722  is set to be 10 um/s, a large number of motile sperms can be collected; when the maximum velocity of the flow field  722  is set to be 30 um/s, a lesser number of motile sperms can be collected as compared with the case of the flow field velocity of 10 um/s; when the maximum velocity of the flow field  722  is set to be 50 um/s, a further lesser number of motile sperms can be collected while the sperm motility of the collected sperms in this case is stronger. In other words, the number of the sperms collected at the end  714   a  that have sufficient motility to overcome the background velocity decreases with the increase of the velocity of the flow field  722 . In addition, in one embodiment, an observation device  730 , for example, a microscope and a charge coupled device (CCD), can further be provided at the end  714   a  to observe the morphology of the collected sperms. Because the collected sperms are able to move against the background flow field  722  toward the junction  710 , the sperm motility of the separated sperms can be evaluated based on the set velocity of the flow field  722 . 
     The velocity of the flow field  722 ,  724  and  726  can be adjusted by changing the height of liquid in the reservoirs  706   a ,  706   b  and  706   c  to generate different hydrostatic pressure or by modifying the width of the microfluidic regions  712 ,  714  and  716 . In one embodiment, the height of liquid in the reservoir  706   c  is greater than the height of liquid in the reservoirs  706   a  and  706   b , and, therefore, the buffer solution in the reservoir  706   c  can establish the flow fields  722 ,  724  with opposite directions in the microfluidic regions  712 ,  714 , respectively. 
     While the microfluidic region  716  is illustrated as being connected to the microfluidic regions  712  and  714  at a right angle in the embodiment of  FIGS. 7A and 7B , it is to be understood that this is for the purposes of illustration only and therefore should not be regarded as limiting. Rather, in other embodiments, the microfluidic region  716  may also be connected to the junction  710  at an angle not perpendicular to the extending direction of the microfluidic regions  712  and  714 . 
     In addition, in another embodiment, the microfluidic region  714  of  FIG. 7A  and  FIG. 7B  may also be configured to include a shrunk portion  120  of  FIG. 1B  and a detector disposed at the shrunk portion as a detecting region (not shown). The shrunk portion of the microfluidic region  714  is, for example, parallel to the extending direction of the microfluidic region  714  and includes an extending channel having a specific length. Besides, the shrunk portion of the microfluidic region  714  may be disposed adjacent a connecting area of the microfluidic region  716  and the microfluidic region  714 , or disposed between the junction  710  and the end  714   a . Therefore, the present invention is not intended to limit the shrunk portion to any particular position described herein. As such, in addition to separating and collecting sperms with different motility by varying the background velocity of the flow field  722 , the present biochip system can also determine the sperm quality of the sperms that are screen out by passing through the shrunk portion with the detector disposed at the microfluidic region  714 . Determining the sperm quality of each sperm passing through the shrunk portion of the microfluidic region  714  using the detector may be performed in the manner similar to that illustrated in  FIG. 2A  and  FIG. 2C  and therefore is not repeated herein. 
     While three microfluidic regions with different flow fields are foamed on the upper surface of the substrate in the embodiment of  FIG. 7A  and  FIG. 7B , it is noted that the microfluidic region  716  may be replaced with another element that can provide a high velocity flow field, which is described below with reference to  FIG. 7C .  FIG. 7C  is a top view of a biochip system according to a ninth embodiment of the present invention, wherein elements that are the same as in  FIG. 7A  and  FIG. 7B  are referenced by the same numerals and explanation thereof is not repeated herein. 
     Referring to  FIG. 7C , only two microfluidic regions  712 ′ and  714 ′ in communication with each other are formed on the upper surface of a substrate  702  of a biochip system  700 ′. The microfluidic region  712 ′ and the microfluidic region  714 ′ extend in the same direction and meet at a junction  710 ′. In addition, the microfluidic region  716 ′ connected to the junction  710 ′ is disposed above the substrate  702  and is not located on the plane on which the microfluidic regions  712 ′ and  714 ′ are located. The microfluidic region  716 ′ is, for example, an element capable of providing a high velocity flow field, such as, an injector that injects the buffer solution into the microfluidic regions  712 ′ and  714 ′ from above the junction  710 ′. 
     When the buffer solution is injected from the microfluidic region  716 ′ to the junction  710 ′, it forms a flow field  722 ′ in the microfluidic region  712 ′ and a flow field  724 ′ in the microfluidic region  714 ′. Because the direction of the flow field  722 ′ is opposite to the direction of the flow field  724 ′, the biochip system  700 ′ can also provide flow fields similar to that shown in  FIG. 7B . When a semen sample is loaded at the end of the microfluidic region  712 ′, if a sperm is able to overcome the velocity of the flow field  722 ′ to move upstream toward the junction  710 ′, the sperm can be rapidly carried to the collecting end by the high velocity flow field  724 ′ after passing through the junction  710 ′. 
     In order to verify the biochip system is indeed capable of effectively separating sperms with specific motility, several experiments are conducted which will now be described below. It is to be understood that this is for the purposes of illustrating the sperm separating results under different flow field configurations of the biochip system and should not be regarded as limiting. 
     Experiments 
       FIG. 8  is a comparison diagram of the percentage of motile sperm in a semen sample prior to and after a separation process using the biochip system  700  of  FIG. 7A  and  FIG. 7B . 
     As shown in  FIG. 8 , the experiments use four different semen samples. These semen samples are loaded at the ends  712   a  of the microfluidic regions  712  with the maximum flow field velocity of 10 um/s, 30 um/s, and 50 um/s, respectively, and sperms capable of overcoming different background flow field velocity to move upstream are collected. The sperm collecting ends 20 minutes later. The percentage of motile sperms in the semen collected at respective collecting ends with different flow field velocity, together with the percentage of sperms in the initial semen sample without undergoing any separation processing, are then plotted in the comparison diagram of  FIG. 8 . From the comparison it can be apparent that the percentage of motile sperm in the semen samples undergoing the separation process at three maximum velocities using the biochip system of the present invention is much greater than the percentage of motile sperm in the initial unprocessed semen sample. Furthermore, the percentage of motile sperm after the semen undergoes the separation process is close to 100%, which means the separated sperms almost all have a certain level of motility. 
     In summary, the biochip system of the present invention employs the microfluidics technology to design the flow field such that sperms in the semen sample can overcome the background velocity to move upstream and the sperm number and concentration of sperms that move upstream a fixed distance within a fixed time period can be detected, thus facilitating evaluating the sperm motility. In addition, the biochip system of the present invention is capable of screening out or separating the sperms with different specific motility by controlling the velocity of the background flow field. 
     Besides, in the method for determining sperm quality and separating sperms, a simple structure is used to generate desired flow fields in the microfluidic regions to determine the sperm concentration of the sperms capable of moving upstream to further evaluate the sperm motility and collect sperms with a certain level of motility. Moreover, the semen sample does not need to undergo any preprocessing such as dyeing process, marking process, or centrifuging process. Therefore, the biochip system of the present invention is capable of rapidly determining the sperm quality and evaluating the sperm motility in a simplified manner, and further separating and collecting sperms with different motility by controlling the background flow field velocity. 
     It will be apparent to those skilled in the art that various modifications and variations can be made to the structure of the present invention without departing from the scope or spirit of the invention. In view of the foregoing, it is intended that the present invention cover modifications and variations of this invention provided they fall within the scope of the following claims and their equivalents.