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).
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.
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.
Referring to
In addition to the material layer 104, other components, such as reservoirs 106a, 106b and 106c, can be formed on the substrate 102. The reservoirs 106a, 106b and 106c 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 106a, 106b and 106c can be used to store or collect samples, reagents or buffer solutions.
In the area 110 shown in
The microfluidic region 112 has an end 112a positioned at a side opposite to the junction 118. The end 112a acts, for example, as a semen sample loading end and communicates with the reservoir 106a. The reservoir 106a contains, for example, a semen sample that does not undergo any preprocessing. The length L1 of the microfluidic region 112 is about within the range from 0.05 mm to 40 mm, and the width W1 of the microfluidic region 112 is about within the range from 5 um to 10000 um. The microfluidic region 114 has an end 114a positioned at a side opposite to the junction 118. The end 114a acts, for example, as an exit end for moving sperms and communicates with the reservoir 106b. The reservoir 106b contains, for example, RPMI 1640 nutrient solution. The length L2 of the microfluidic region 114 is about within the range from 0.01 mm to 40 mm, and the width W2 of the microfluidic region 114 is about within the range from 5 um to 10000 um. The microfluidic region 116 has an end 116a positioned at a side opposite to the junction 118. The end 116a acts, for example, as a flow field source end to provide a buffer solution and communicates with the reservoir 106c. The reservoir 106c 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 L3 of the microfluidic region 116 is about within the range from 0.1 mm to 40 mm, and the width W3 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 LA. The shrunk portion 120, acting as a detecting region, has a smaller conduit width WA 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 WA 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
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 116a 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 116a to the junction 118, the buffer solution is separated into two parts, one of which flows from the junction 118 to the end 112a 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 114a 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 112a 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 106a, 106b and 106c 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 106c is greater than the height of liquid in the reservoir 106b and, therefore, the buffer solution in the reservoir 106c 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
The detector 200 used in
Referring to
After measuring for a specific time period (t=t0), as shown in
As shown in
For example, as shown in
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 114a 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 114a. The collecting portion 302 may also be the reservoir 106b of
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.
As shown in
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 402a 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 LA 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.
In the fourth embodiment, the main elements of the biochip system of
Similarly, the detector 200 used in
Referring to
After measuring for a specific time period (t=t0), as shown in
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.
Referring to
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 602a and the part of microfluidic region 604 adjacent the end 604a 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
In addition, the present invention is not intended to limit the number of the microfluidic regions to any particular number described herein. Referring to
While the biochip system is illustrated as having two microfluidic regions connected to the semen sample loading end in
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.
Referring to
Referring to
The microfluidic region 712 has an end 712a acting, for example, as a semen sample loading end and communicating with the reservoir 706a. The reservoir 706a contains, for example, a semen sample that does not undergo any preprocessing. The length L4 of the microfluidic region 712 is about within the range from 0.05 mm to 40 mm, and the width W4 of the microfluidic region 712 is about within the range from 5 um to 10000 um. The microfluidic region 714 has an end 714a acting, for example, as an exit end for moving sperms and communicating with the reservoir 706b. The reservoir 706b contains, for example, RPMI 1640 nutrient solution. The length L5 of the microfluidic region 714 is about within the range from 0.01 mm to 40 mm, and the width W5 of the microfluidic region 714 is about within the range from 10 um to 10000 um. The microfluidic region 716 has an end 716a acting, for example, as a flow field source end to provide a buffer solution and communicating with the reservoir 706c. The reservoir 706c 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 L6 of the microfluidic region 716 is about within the range from 0.01 mm to 40 mm, and the width W6 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
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 714a 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 714a 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 706a, 706b and 706c 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 706c is greater than the height of liquid in the reservoirs 706a and 706b, and, therefore, the buffer solution in the reservoir 706c 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
In addition, in another embodiment, the microfluidic region 714 of
While three microfluidic regions with different flow fields are foamed on the upper surface of the substrate in the embodiment of
Referring to
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
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.
As shown in
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.
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.
Number | Date | Country | |
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61276529 | Sep 2009 | US |