TW202016285A

TW202016285A - Integrated chip and method for sperm sorting, oocyte incubation and in vitro fertilization

Info

Publication number: TW202016285A
Application number: TW107137113A
Authority: TW
Inventors: 曾繁根; 曾詠欽; 陳約任; 張權
Original assignee: 國立清華大學
Priority date: 2018-10-22
Filing date: 2018-10-22
Publication date: 2020-05-01
Also published as: TWI710630B

Abstract

An integrated chip is provided and includes a sperm sorting system, an oocyte incubation and fertilization system and a liquid perfusion system. The sperm sorting system includes an inlet, an outlet, a main channel and a branch channel. The main channel has a width which increases along a direction from the inlet toward the outlet. The branch channel is connected with the main channel. The oocyte incubation and fertilization system is connected with the branch channel and has a first hole part and a second hole part. The diameter of the first hole is larger than an average diameter of the oocyte to hold the oocyte to be fertilized by the sperm. The second hole part is disposed below the first hole part, and the diameter of the second hole is smaller than the oocyte. The selected sperm is introduced to the oocyte incubation and fertilization system through the branch channel, to fertilize with the oocyte in the first hole. The liquid perfusion system is disposed below the second hole part and a liquid exchange is performed onto the incubation and fertilization system through the second hole part.

Description

用於***分選、卵子培育以及體外受精的整合式晶片及方法Integrated chip and method for sperm sorting, egg cultivation and in vitro fertilization

本發明是有關於一種系統及方法，且特別是有關於一種用於***分選、卵子培育以及體外受精的整合式晶片及方法。The invention relates to a system and method, and in particular to an integrated chip and method for sperm sorting, egg cultivation and in vitro fertilization.

根據資料統計，台灣地區育齡期夫妻中不孕之比例約為10~15%，面臨如此高的不孕症比例，體外受精技術（in vitro fertilization（IVF），即一般所通稱的「試管嬰兒」）的成功懷孕率卻只有33％，如此低的成功率，大多歸咎於執行傳統試管嬰兒技術時的檢體處理方式不夠溫和或因為人為失誤，導致檢體健康狀況大幅衰退甚至死亡，若能開發一系統可以以更溫和的方式處理檢體，相信對體外授精技術的成功率能夠有顯著的提升。According to statistics, the ratio of pregnancy in couples of childbearing age in Taiwan is about 10-15%, facing such a high ratio of pregnancy 例, in vitro fertilization (IVF), commonly known as "test tube baby" ) The successful pregnancy rate is only 33%. Such a low success rate is mostly due to the fact that the sample processing method when performing traditional IVF techniques is not gentle enough or due to human error, which leads to a significant decline in the health of the sample or even death. If it can be developed A system can process specimens in a more gentle manner, and I believe that the success rate of in vitro fertilization technology can be significantly improved.

本發明提供一種整合式晶片，用於***分選、卵子培育以及體外受精。The invention provides an integrated chip for sperm sorting, egg cultivation and in vitro fertilization.

本發明另提供一種方法，用於***分選、卵子培育以及體外受精。The invention also provides a method for sperm sorting, egg cultivation and in vitro fertilization.

本發明的用於***分選、卵子培育以及體外受精的整合式晶片包括***分選系統、卵子培育與精卵結合系統以及液體灌注系統。***分選系統包括***入口部、***出口部、主流道以及分支流道。主流道與***入口部及***出口部連通，主流道具有由***入口部朝向***出口部逐漸增大的寬度。分支流道位於***入口部與***出口部之間且與主流道連通。卵子培育與精卵結合系統與分支流道連通，包括第一貫孔部與第二貫孔部。第一貫孔部包括多個第一貫孔，第一貫孔的孔徑大於卵子的平均直徑，用以容納待與***結合的卵子。第二貫孔部位於第一貫孔部下方且包括多個第二貫孔，第二貫孔的孔徑小於卵子，用以進行液體交換。經***分選系統分選後的***透過分支流道被導入卵子培育與精卵結合系統，以與位於第一貫孔中的卵子結合。液體灌注系統包括液體入口部、液體出口部以及液體流道。液體流道位於液體入口部及液體出口部之間且與液體入口部及液體出口部連通。液體流道的一部分位於第二貫孔部下方且通過第二貫孔部與卵子培育與精卵結合系統進行液體交換。The integrated wafer for sperm sorting, egg cultivation and in vitro fertilization of the present invention includes a sperm sorting system, an egg cultivation and sperm and egg combination system, and a liquid perfusion system. The sperm sorting system includes a sperm inlet, a sperm outlet, a main flow channel, and a branch flow channel. The main flow path communicates with the sperm entrance and sperm exit. The main flow path has a width that gradually increases from the sperm entrance to the sperm exit. The branch flow channel is located between the sperm inlet and the sperm outlet and communicates with the main channel. The egg cultivation and sperm-egg combination system communicates with the branch flow channel, and includes a first through-hole portion and a second through-hole portion. The first through-hole portion includes a plurality of first through-holes, and the diameter of the first through-hole is larger than the average diameter of the egg to accommodate the egg to be combined with sperm. The second through-hole portion is located below the first through-hole portion and includes a plurality of second through-holes. The diameter of the second through-hole is smaller than that of the egg for liquid exchange. The sperm sorted by the sperm sorting system is introduced into the egg cultivating and sperm-egg combining system through the branch flow channel to combine with the egg located in the first through hole. The liquid perfusion system includes a liquid inlet, a liquid outlet, and a liquid flow path. The liquid flow path is located between the liquid inlet and the liquid outlet and communicates with the liquid inlet and the liquid outlet. A part of the liquid flow path is located below the second through-hole portion and exchanges liquid through the second through-hole portion and the egg breeding and sperm-egg combination system.

在本發明的一實施例中，上述的第一貫孔部與第二貫孔部的材料包括聚二甲基矽氧烷（PDMS）。In an embodiment of the present invention, the materials of the first and second through-hole portions include polydimethylsiloxane (PDMS).

在本發明的一實施例中，上述的第一貫孔是陣列排列。In an embodiment of the invention, the above-mentioned first through holes are arranged in an array.

在本發明的一實施例中，上述的第二貫孔是陣列排列。In an embodiment of the invention, the above-mentioned second through holes are arranged in an array.

在本發明的一實施例中，上述的第二貫孔的孔徑大於30um。In an embodiment of the invention, the diameter of the second through hole is greater than 30um.

在本發明的一實施例中，上述的主流道包括第一區段及與第一區段連接的第二區段，第一區段位於***入口部與第二區段之間，第二區段位於第一區段與***出口部之間，其中第一區段具有實質上相同的寬度，第二區段具有逐漸增大的寬度且其最小寬度大於第一區段的寬度。In an embodiment of the invention, the main flow path includes a first section and a second section connected to the first section. The first section is located between the sperm inlet and the second section. The second section The segment is located between the first segment and the sperm outlet, where the first segment has substantially the same width, the second segment has a gradually increasing width and its minimum width is greater than the width of the first segment.

在本發明的一實施例中，上述的分支流道的寬度小於第二區段的寬度。In an embodiment of the invention, the width of the aforementioned branch flow channel is smaller than the width of the second section.

在本發明的一實施例中，上述的***分選系統更包括阻擋結構，阻擋結構位於所述主流道中且鄰近於所述***出口部，且阻擋結構與主流道的側壁之間具有距離。In an embodiment of the present invention, the above-mentioned sperm sorting system further includes a blocking structure located in the main channel and adjacent to the sperm outlet, and there is a distance between the blocking structure and the side wall of the main channel.

在本發明的一實施例中，上述的阻擋結構為啞鈴形。In an embodiment of the invention, the above-mentioned blocking structure is dumbbell-shaped.

在本發明的一實施例中，上述的***入口部與***出口部為槽體。In an embodiment of the invention, the sperm inlet and the sperm outlet are grooves.

在本發明的一實施例中，上述的液體流道具有液體交換區，位於卵子培育與精卵結合系統下方且具有與第二貫孔部相符的輪廓。In an embodiment of the present invention, the above-mentioned liquid flow channel has a liquid exchange area, which is located below the egg breeding and sperm-egg combination system and has a contour corresponding to the second through hole portion.

在本發明的一實施例中，包括第一薄膜、位於第一薄膜下方的第二薄膜、位於第二薄膜下方的第三薄膜以及位於第三薄膜下方的第四薄膜。第一薄膜包括對應於***入口部的第一通孔、對應於***出口部的第二通孔、對應於主流道與分支流道的第一溝槽、對應於卵子培育與精卵結合系統的第三通孔、對應於液體入口部的第四通孔以及對應於液體出口部的第五通孔。第二薄膜包括第一貫孔部、與第四通孔對應的第六通孔以及與第五通孔對應的第七通孔，其中位於第一通孔、第二通孔以及第一溝槽下方的第二薄膜分別作為***入口部、***出口部以及主流道與分支流道的底部。第三薄膜包括第二貫孔部、與第六通孔對應的第八通孔以及與第七通孔對應的第九通孔。第四薄膜包括彼此相連的第一凹槽、第二溝槽以及第二凹槽，其中第一凹槽對應於第八通孔且作為液體入口部的底部，第二溝槽作為液體流道，第二凹槽對應於第九通孔且作為液體出口部的底部。In an embodiment of the invention, it includes a first film, a second film under the first film, a third film under the second film, and a fourth film under the third film. The first membrane includes a first through hole corresponding to the sperm inlet, a second through hole corresponding to the sperm outlet, a first groove corresponding to the main flow channel and the branch flow channel, and corresponding to the egg culturing and sperm and egg combination system. The third through hole, the fourth through hole corresponding to the liquid inlet portion, and the fifth through hole corresponding to the liquid outlet portion. The second film includes a first through hole portion, a sixth through hole corresponding to the fourth through hole, and a seventh through hole corresponding to the fifth through hole, wherein the first through hole, the second through hole, and the first trench are located The second thin film below serves as the sperm inlet, sperm outlet, and the bottom of the main channel and branch channel. The third film includes a second through hole portion, an eighth through hole corresponding to the sixth through hole, and a ninth through hole corresponding to the seventh through hole. The fourth film includes a first groove, a second groove, and a second groove connected to each other, wherein the first groove corresponds to the eighth through hole and serves as a bottom of the liquid inlet portion, and the second groove serves as a liquid flow path, The second groove corresponds to the ninth through hole and serves as the bottom of the liquid outlet portion.

在本發明的一實施例中，上述的第一薄膜、第二薄膜、第三薄膜以及第四薄膜的材料包括聚二甲基矽氧烷。In an embodiment of the invention, the materials of the first film, the second film, the third film and the fourth film include polydimethylsiloxane.

在本發明的一實施例中，上述的第一薄膜、第二薄膜、第三薄膜以及第四薄膜中相鄰兩者彼此黏合。In an embodiment of the invention, adjacent ones of the first film, the second film, the third film, and the fourth film are bonded to each other.

本發明的用於***分選、卵子培育以及體外受精的方法包括以下步驟。提供前述的整合式晶片。將含***的樣本液滴入***入口部中，使得***分選系統對含***的樣本液進行***分選。將含卵子的樣本液滴入卵子培育與精卵結合系統中，使得卵子分別位於第一貫孔中。使經分選的***通過分支流道被導入卵子培育與精卵結合系統，以於第一貫孔中與卵子結合成受精卵。The method for sperm sorting, egg cultivation and in vitro fertilization of the present invention includes the following steps. The aforementioned integrated chip is provided. The sperm-containing sample is dropped into the sperm inlet, so that the sperm sorting system performs sperm sorting on the sperm-containing sample liquid. Drop the egg-containing sample into the egg cultivating and sperm-egg combination system, so that the eggs are located in the first through holes respectively. The sorted sperm is introduced into the egg cultivation and sperm and egg combination system through the branch flow channel to combine with the egg to form a fertilized egg in the first through hole.

在本發明的一實施例中，更包括在將含卵子的樣本液滴入卵子培育與精卵結合系統中之後，對卵子進行透明帶去除步驟。In an embodiment of the present invention, the method further includes the step of removing the zona pellucida after the egg-containing sample droplet is dropped into the egg cultivation and sperm-egg combination system.

在本發明的一實施例中，更包括在將含卵子的樣本液滴入卵子培育與精卵結合系統中之後，密封卵子培育與精卵結合系統，以及在***分選系統對含***的樣本液完成***分選之後，通過打開卵子培育與精卵結合系統使得經分選的***通過分支流道進入卵子培育與精卵結合系統。In an embodiment of the present invention, it further includes sealing the egg cultivating and sperm-egg combining system after dropping the egg-containing sample into the egg culturing and sperm-egg combining system, and sperm-containing samples in the sperm sorting system After the sperm sorting is completed by the liquid, the egg cultivating and sperm egg combination system is opened so that the sorted sperm enters the egg culturing and sperm egg combination system through the branch flow channel.

在本發明的一實施例中，更包括在將含***的樣本液滴入***入口部中之後，密封***分選系統，以及在將含卵子的樣本液滴入卵子培育與精卵結合系統中之後，通過打開***分選系統以進行***分選。In an embodiment of the present invention, it further includes sealing the sperm sorting system after dropping the sperm-containing sample droplets into the sperm inlet, and after dropping the egg-containing sample droplets into the egg culturing and sperm-egg combination system After that, sperm sorting is performed by turning on the sperm sorting system.

在本發明的一實施例中，更包括在卵子培育與精卵結合系統中培養受精卵。In an embodiment of the present invention, the method further includes cultivating fertilized eggs in an egg cultivating and sperm-egg combination system.

在本發明的一實施例中，更包括使液體在液體灌注系統中流動，其中液體經通過第二貫孔部與卵子培育與精卵結合系統中的液體進行液體交換。In an embodiment of the present invention, the method further includes flowing liquid in the liquid perfusion system, wherein the liquid exchanges liquid with the liquid in the egg cultivating and sperm-egg combining system through the second through-hole portion.

基於上述，本發明將***分選系統、卵子培育與精卵結合系統以及液體灌注系統整合在單一晶片上，使得經***分選系統分選後的高效能***能直接被導入卵子培育與精卵結合系統，以與經培育的卵子原位結合成受精卵，進而達成體外受精的目的。由於本發明的晶片是被動式地分選***、處理卵子以及形成受精卵，故過程溫和而能避免傷害精卵，使得精卵存活率與結合率大幅提升。因此，本發明的晶片不僅能縮短傳統人工生殖技術之操作時間，更透過簡化操作流程來避免因操作時間冗長、人為操作失當而導致的檢體死亡，進而大幅地提升人工生殖技術的成功率。Based on the above, the present invention integrates the sperm sorting system, egg cultivation and sperm-egg combination system, and liquid perfusion system on a single chip, so that the high-performance sperm sorted by the sperm sorting system can be directly introduced into egg cultivation and sperm eggs Combining the system to combine with the cultivated egg in situ to form a fertilized egg, thus achieving the purpose of in vitro fertilization. Since the chip of the present invention passively sorts sperm, processes eggs and forms fertilized eggs, the process is gentle and can avoid sperm egg damage, so that the sperm egg survival rate and binding rate are greatly improved. Therefore, the chip of the present invention can not only shorten the operation time of the traditional artificial reproduction technology, but also avoid the death of the specimen caused by the lengthy operation time and the artificial improper operation by simplifying the operation process, thereby greatly improving the success rate of the artificial reproduction technology.

為讓本發明的上述特徵和優點能更明顯易懂，下文特舉實施例，並配合所附圖式作詳細說明如下。In order to make the above-mentioned features and advantages of the present invention more obvious and understandable, the embodiments are specifically described below in conjunction with the accompanying drawings for detailed description as follows.

為了讓本發明之上述及其他目的、特徵、優點能更明顯易懂，下文將特舉本發明較佳實施例，並配合所附圖式，作詳細說明如下。再者，本發明所提到的方向用語，例如「上」、「下」、「前」、「後」、「左」、「右」、「內」、「外」或「側面」等，僅是參考附加圖式的方向。因此，使用的方向用語是用以說明及理解本發明，而非用以限制本發明。In order to make the above and other objects, features, and advantages of the present invention more comprehensible, the preferred embodiments of the present invention will be specifically described below in conjunction with the accompanying drawings, which will be described in detail below. Furthermore, the directional terms mentioned in the present invention, such as "upper", "lower", "front", "rear", "left", "right", "inner", "outer" or "side", etc., Just refer to the directions of the attached drawings. Therefore, the directional terminology is used to illustrate and understand the present invention, not to limit the present invention.

本發明利用漸擴型流道搭配***會逆流的天性，透過漸進式地增加流道某部份的截面積來階段性地減少流道的流速，藉此能夠階段性地對不同活動能力的***進行區隔。具體而言，較有活力的***將能抵抗流體（即樣本液中的流體）而留在原位或逆流而上，而較不具活力的***將被流體帶往出口部。另一方面，本發明還透過使用阻擋結構來阻擋高活動力***，而將活動力不足或死亡的***帶往出口部。更重要的是，本發明透過設置分支流道，使得分選後的***能通過分支流道被導入卵子培育與精卵結合系統中。The present invention utilizes the nature of the progressive flow channel and the sperm will counterflow, and gradually reduces the flow velocity of the flow channel by gradually increasing the cross-sectional area of a certain part of the flow channel, thereby being able to stage sperm with different mobility in stages Make a segmentation. Specifically, the more viable sperm will be able to resist the fluid (ie, the fluid in the sample fluid) and remain in place or upstream, while the less viable sperm will be carried by the fluid to the outlet. On the other hand, the present invention also uses the blocking structure to block high-motility sperm, and brings the sperm with insufficient mobility or death to the outlet. More importantly, in the present invention, by setting a branch flow channel, the sorted sperm can be introduced into the egg breeding and sperm-egg combination system through the branch flow channel.

圖1A是依照本發明的實施例的一種整合式晶片的立體示意圖，圖1B是圖1A的整合式晶片的***示意圖，圖1C是圖1A的整合式晶片的***分選系統的仰視圖，以及圖1D是圖1A的整合式晶片的剖面示意圖。請同時參照圖1A至圖1D，整合式晶片10包括***分選系統100、卵子培育與精卵結合系統200以及液體灌注系統300。1A is a schematic perspective view of an integrated chip according to an embodiment of the present invention, FIG. 1B is an exploded schematic view of the integrated chip of FIG. 1A, FIG. 1C is a bottom view of the sperm sorting system of the integrated chip of FIG. 1A, and FIG. 1D is a schematic cross-sectional view of the integrated chip of FIG. 1A. Please refer to FIGS. 1A to 1D at the same time. The integrated wafer 10 includes a sperm sorting system 100, an egg cultivating and sperm-egg combining system 200, and a liquid perfusion system 300.

***分選系統100包括***入口部102、***出口部104、主流道106以及分支流道108。在本實施例中，***入口部102用於接收含有***的樣本。***出口部104用於接收流出主流道106的液體。在本實施例中，***入口部102與***出口部104例如分別是槽體。***入口部102與***出口部104的直徑例如是1.8mm至2.2mm，且例如是2mm。***入口部102與***出口部104的深度例如是1.8mm至2.2mm，且例如是2mm。主流道106與分支流道108例如分別是具有深度的溝槽。主流道106位於***入口部102及***出口部104之間且連通兩者。The sperm sorting system 100 includes a sperm inlet 102, a sperm outlet 104, a main flow path 106, and a branch flow path 108. In this embodiment, the sperm inlet 102 is used to receive a sample containing sperm. The sperm outlet 104 is used to receive the liquid flowing out of the main channel 106. In this embodiment, the sperm inlet 102 and the sperm outlet 104 are, for example, tanks. The diameters of the sperm inlet portion 102 and the sperm outlet portion 104 are, for example, 1.8 mm to 2.2 mm, and for example, 2 mm. The depth of the sperm inlet 102 and the sperm outlet 104 is, for example, 1.8 mm to 2.2 mm, and for example, 2 mm. The main channel 106 and the branch channel 108 are, for example, deep grooves. The main flow channel 106 is located between and connects the sperm inlet 102 and the sperm outlet 104.

在應用時，包含***的樣本液例如是首先地被放置於***入口部102處，接著由於***入口部102與主流道106中的液體高度的差異，樣本會由***入口部102進入主流道106。其中，樣本中較有活力的***將能抵抗流體而維持原位或逆流而上，而樣本中較不具活力的***將被流體帶至***出口部104。In application, the sample liquid containing sperm is first placed at the sperm inlet 102, for example, due to the difference in liquid height between the sperm inlet 102 and the mainstream channel 106, the sample will enter the mainstream channel 106 from the sperm inlet 102 . Among them, the more active sperm in the sample will be able to resist the fluid to maintain in situ or upstream, while the less active sperm in the sample will be brought to the sperm outlet 104 by the fluid.

在本實施例中，主流道106例如是設置有彼此連接的第一區段106A、第二區段106B以及第三區段106C，其為用於對***進行分類與分選之區段。第一區段106A位於***入口部102與第二區段106B之間，具有實質上相同的寬度，且此寬度小於或等於第二區段106B的最小寬度。第二區段106B位於第一區段106A與第三區段106C之間，具有沿著由***入口部102朝向***出口部104方向逐漸增大的寬度，因此又可稱為漸擴式區段。由於流體的流速與其通過的通道的大小是成反比，故此，漸擴式區段得透過不同速度的流體來區隔多種具有不同活動能力的***並使其停留於相對應的區段中。更明確地說，漸擴式區段相對於***入口部102方向的部分之垂直切面面積中可供流體流動部份的面積是較漸擴式區段相對於***出口部104方向的部分小。本設計所產生的流體流速可透過流體力學的公式計算。其中具有活動力的***會抵抗流體速度，依據***活動能力不同停留在不同流體速度的位置。需知悉的是，前述的垂直切面面積是指垂直於本發明的***分選系統100的延伸長度方向的垂直切面。更明確的說，必要時，垂直切面的面積可選擇性地被解釋為流體得自垂直切面流通之面積的大小，而定義***分選系統100的材料部分所佔用之面積應得予以忽略。In this embodiment, the main flow channel 106 is, for example, a first segment 106A, a second segment 106B, and a third segment 106C connected to each other, which are segments for sorting and sorting sperm. The first section 106A is located between the sperm inlet 102 and the second section 106B and has substantially the same width, and this width is less than or equal to the minimum width of the second section 106B. The second section 106B is located between the first section 106A and the third section 106C, and has a width that gradually increases in the direction from the sperm inlet 102 to the sperm outlet 104, so it can also be called a gradually expanding section . Since the flow velocity of the fluid is inversely proportional to the size of the channel through which it passes, therefore, the gradually expanding section has to separate multiple sperms with different mobility and stay in the corresponding section through fluids of different speeds. More specifically, the area of the section of the vertical section of the gradually expanding section relative to the direction of the sperm inlet 102 is smaller than the portion of the gradually expanding section relative to the direction of the sperm outlet 104. The fluid flow rate generated by this design can be calculated by the formula of fluid mechanics. The sperm with mobility will resist the fluid velocity, and stay at different fluid velocity positions according to the sperm mobility. It should be noted that the aforementioned vertical cross-sectional area refers to a vertical cross-section perpendicular to the extending length direction of the sperm sorting system 100 of the present invention. More specifically, when necessary, the area of the vertical section can be selectively interpreted as the size of the area through which the fluid flows from the vertical section, and the area occupied by the material portion that defines the sperm sorting system 100 should be ignored.

再者，為了阻擋高活動力***，本發明的***分選系統100的主流道106中又選擇性地增設有第三區段106C，因此第三區域106C又稱為阻擋區域。第三區段106C位於第二區段106B與***出口部104之間，為主流道106中鄰近於***出口部104的區段。在本實施例中，第三區段106C例如是具有實質上相同的寬度，且此寬度大於或等於第二區段106B的最大寬度。於第三區段106C中，流體例如是以相較於指定流速少例如三倍之流速流經處，唯其不以三倍為限，第三區段106C之流速可為指定流速的三倍至二十倍之間。在本實施例中，第三區段106C中例如是設置有阻擋結構110，阻擋結構110例如是用於減少***分選系統100之第三區段106C中可供流體流動的空間進而增加流體流經第三區段106C時的流速。在本實施例中，阻擋結構110例如是設置在第二區段106B與第三區段106C的交界處。在本實施例中，阻擋結構110例如是呈一啞鈴形，而呈啞鈴形的阻擋結構110例如是具有頭端110A、連接端110B以及末端110C，連接端110B例如是連接並位於頭端110A及末端110C之間，頭端110A及末端110C與第三區段106C之側壁之間具有一距離d，此距離d例如是大於或等於50μm。頭端110A、連接端110B以及末端110C之排列方向例如是與流體之流動方向相互垂直，以使流體可自阻擋結構110相對於連接端110B的另一方向之兩端往後流通。Furthermore, in order to block high-activity sperm, a third section 106C is selectively added to the main channel 106 of the sperm sorting system 100 of the present invention, so the third region 106C is also called a blocking region. The third section 106C is located between the second section 106B and the sperm outlet 104 and is the section adjacent to the sperm outlet 104 in the main channel 106. In this embodiment, the third section 106C has, for example, substantially the same width, and this width is greater than or equal to the maximum width of the second section 106B. In the third section 106C, for example, the fluid flows at a flow rate that is less than, for example, three times the specified flow rate, but it is not limited to three times. The flow rate of the third section 106C can be three times the specified flow rate Between twenty times. In this embodiment, a blocking structure 110 is provided in the third section 106C, for example, the blocking structure 110 is used to reduce the space available for fluid flow in the third section 106C of the sperm sorting system 100 to increase the fluid flow. Flow rate through the third section 106C. In this embodiment, the blocking structure 110 is disposed at the junction of the second section 106B and the third section 106C, for example. In this embodiment, the blocking structure 110 is, for example, a dumbbell shape, and the dumbbell-shaped blocking structure 110 has, for example, a head end 110A, a connecting end 110B, and an end 110C. The connecting end 110B is, for example, connected and located at the head end 110A and There is a distance d between the tip 110C, the tip 110A and the tip 110C, and the side wall of the third section 106C. The distance d is, for example, greater than or equal to 50 μm. The arrangement direction of the head end 110A, the connection end 110B, and the end 110C is, for example, perpendicular to the flow direction of the fluid, so that the fluid can circulate backward from both ends of the blocking structure 110 relative to the connection end 110B in the other direction.

在本實施例中，阻擋結構110之頭端110A、連接端110B以及末端110C例如是圍設有一用於儲存***的集精槽110D，集精槽110D具有一深度，深度的延伸方向例如是與流體之流動方向相同。透過集精槽110D之設計，其可以有效阻隔死的與活的***。在本實施例中，主流道106於第三區段106C中的平均垂直切面面積例如為第二區段106B中與第三區段106C連接處的區段之三分之一至二十分之一間，藉此，第三區段106C之流速將得以例如被提升至三倍至二十倍之間。In this embodiment, the head end 110A, the connecting end 110B, and the end 110C of the blocking structure 110 are surrounded by a sperm collecting tank 110D for storing sperm. The sperm collecting tank 110D has a depth. The flow direction of the fluid is the same. Through the design of sperm collection tank 110D, it can effectively block dead and live sperm. In this embodiment, the average vertical cross-sectional area of the main channel 106 in the third section 106C is, for example, one-third to two-twentieth of the section where the third section 106C is connected to the third section 106B One, by this, the flow velocity of the third section 106C will be increased, for example, between three times and twenty times.

分支流道108與主流道106連通，且用以連接***分選系統100與卵子培育與精卵結合系統200。在本實施例中，經***分選系統100分選後的高效能***會逆流至分支流道108與主流道106之交界處，而被動式地被推進分支流道108中，進而被導入卵子培育與精卵結合系統200。在本實施例中，分支流道108例如是連接於第二區段106B的前端，也就是樣本液由第一區段106A進入至第二區段106B的區域。分支流道108之延伸方向例如是垂直於由***入口部102至***出口部104之延伸方向。The branch flow channel 108 communicates with the main flow channel 106 and is used to connect the sperm sorting system 100 and the egg cultivation and sperm and egg combination system 200. In this embodiment, the high-performance sperm sorted by the sperm sorting system 100 will flow back to the junction of the branch flow channel 108 and the main flow channel 106, and will be passively pushed into the branch flow channel 108, and then introduced into the egg breeding Combination system 200 with sperm and egg. In this embodiment, the branch flow channel 108 is, for example, connected to the front end of the second section 106B, that is, the area where the sample liquid enters the second section 106B from the first section 106A. The extending direction of the branch flow channel 108 is, for example, perpendicular to the extending direction from the sperm inlet 102 to the sperm outlet 104.

在本實施例中，分支流道108例如是具有實質上相同的寬度，且此寬度例如是小於主流道106的最小寬度，即小於第一區段106A的寬度。在一實施例中，分支流道108的寬度例如是40μm至60μm，且例如是50μm，而第一區段106A的寬度例如是80μm至120μm，且例如是100μm。在一實施例中，分支流道108的長度例如是1.8mm至2.2mm，且例如是2mm，而主流道106的長度例如是8mm至10mm，且例如是12mm。在本實施例中，主流道106與分支流道108的深度例如是相同，例如是80μm至120μm。在一實施例中，主流道106與分支流道108的深度例如是100μm。In this embodiment, the branch flow channel 108 has, for example, substantially the same width, and the width is, for example, smaller than the minimum width of the main channel 106, that is, smaller than the width of the first section 106A. In an embodiment, the width of the branch flow channel 108 is, for example, 40 μm to 60 μm, and for example, 50 μm, and the width of the first section 106A is, for example, 80 μm to 120 μm, and for example, 100 μm. In an embodiment, the length of the branch flow channel 108 is, for example, 1.8 mm to 2.2 mm, and for example, 2 mm, and the length of the main flow channel 106 is, for example, 8 mm to 10 mm, and for example, 12 mm. In this embodiment, the depths of the main flow channel 106 and the branch flow channel 108 are, for example, the same, for example, 80 μm to 120 μm. In an embodiment, the depth of the main flow channel 106 and the branch flow channel 108 is, for example, 100 μm.

卵子培育與精卵結合系統200包括槽體202以及配置於槽體202中的第一貫孔部210與第二貫孔部220。槽體202與分支流道108連通。槽體202的直徑例如是2.5mm至3.5mm，且例如是3mm，槽體202的深度例如是1.8mm至2.2mm，且例如是2mm。第一貫孔部210設置於槽體202中。第一貫孔部210包括多個第一貫孔212，第一貫孔212的孔徑大於卵子的平均直徑，用以容納待與***結合的卵子。也就是說，經***分選系統100分選後的***透過分支流道108被導入卵子培育與精卵結合系統200，以與位於第一貫孔212中的卵子結合。在本實施例中，第一貫孔212的孔徑經設計，使得第一貫孔212實質上僅容納一個卵子，如此有利於有效受精卵的形成且有利於單一目標細胞的後續追蹤。在本實施例中，第一貫孔部210例如是由聚二甲基矽氧烷（PDMS）所製成，其中第一貫孔212貫穿聚二甲基矽氧烷。第一貫孔212的孔徑例如是180μm至220μm，且例如是200μm，第一貫孔212的深度例如是180μm至220μm，且例如是200μm。第一貫孔212例如是規則排列，且其最密堆積孔隙率例如是50%至60%。The egg breeding and sperm-egg combination system 200 includes a trough body 202 and a first through-hole portion 210 and a second through-hole portion 220 disposed in the trough body 202. The tank 202 communicates with the branch flow channel 108. The diameter of the groove body 202 is, for example, 2.5 mm to 3.5 mm, and for example, 3 mm, and the depth of the groove body 202 is, for example, 1.8 mm to 2.2 mm, and for example, 2 mm. The first through-hole portion 210 is provided in the groove body 202. The first through-hole portion 210 includes a plurality of first through-holes 212, and the diameter of the first through-hole 212 is larger than the average diameter of the egg to accommodate the egg to be combined with the sperm. That is, the sperm sorted by the sperm sorting system 100 is introduced into the egg cultivating and sperm-egg combining system 200 through the branch flow channel 108 to be combined with the egg located in the first through hole 212. In this embodiment, the diameter of the first through hole 212 is designed so that the first through hole 212 substantially only contains one egg, which is beneficial to the formation of effective fertilized eggs and the subsequent tracking of a single target cell. In the present embodiment, the first through-hole portion 210 is made of, for example, polydimethylsiloxane (PDMS), and the first through-hole 212 penetrates the polydimethylsiloxane. The diameter of the first through hole 212 is, for example, 180 μm to 220 μm, and for example, 200 μm, and the depth of the first through hole 212 is, for example, 180 μm to 220 μm, and for example, 200 μm. The first through holes 212 are, for example, regularly arranged, and the most densely packed porosity is, for example, 50% to 60%.

第二貫孔部220位於第一貫孔部210下方，且第二貫孔部220包括多個第二貫孔222。第二貫孔222的孔徑小於卵子，使得卵子以及受精卵能被固定在第一貫孔212中，因此第二貫孔222可作為槽體202的底部。此外，第二貫孔222能透過壓力差異與其下方的液體灌注系統300進行液體交換，故又稱為液體交換薄膜。在本實施例中，第二貫孔部220例如是由聚二甲基矽氧烷所製成，其中第二貫孔222貫穿聚二甲基矽氧烷。第二貫孔222的孔徑例如是25μm至35μm，且例如是30μm，第二貫孔222的深度例如是50μm至100μm，且例如是75μm。第二貫孔222例如是規則排列，且其最密堆積孔隙率例如是15%至30%。The second through hole portion 220 is located below the first through hole portion 210, and the second through hole portion 220 includes a plurality of second through holes 222. The diameter of the second through hole 222 is smaller than that of the egg, so that the egg and the fertilized egg can be fixed in the first through hole 212, so the second through hole 222 can serve as the bottom of the tank 202. In addition, the second through hole 222 can exchange liquid with the liquid perfusion system 300 under the pressure difference, so it is also called a liquid exchange membrane. In the present embodiment, the second through-hole portion 220 is made of polydimethylsiloxane, for example, and the second through-hole 222 penetrates through the polydimethylsiloxane. The diameter of the second through hole 222 is, for example, 25 μm to 35 μm, and for example, 30 μm, and the depth of the second through hole 222 is, for example, 50 μm to 100 μm, and for example, 75 μm. The second through holes 222 are, for example, regularly arranged, and the densest packing porosity thereof is, for example, 15% to 30%.

液體灌注系統300包括液體入口部302、液體出口部304以及位於液體入口部302與液體出口部304之間且與其連通的液體流道306。在本實施例中，液體入口部302用於接收液體，液體出口部304用於接收流出液體流道306的液體。在本實施例中，液體入口部302與液體出口部304例如分別是槽體。液體入口部302與液體出口部304的直徑例如是1.8mm至2.2mm，且例如是2mm。液體入口部302與液體出口部304的深度例如是1.8mm至2.2mm，且例如是2mm。The liquid perfusion system 300 includes a liquid inlet 302, a liquid outlet 304, and a liquid flow channel 306 located between and in communication with the liquid inlet 302 and the liquid outlet 304. In this embodiment, the liquid inlet portion 302 is used to receive liquid, and the liquid outlet portion 304 is used to receive liquid flowing out of the liquid flow path 306. In this embodiment, the liquid inlet 302 and the liquid outlet 304 are, for example, tanks. The diameters of the liquid inlet portion 302 and the liquid outlet portion 304 are, for example, 1.8 mm to 2.2 mm, and for example, 2 mm. The depth of the liquid inlet portion 302 and the liquid outlet portion 304 is, for example, 1.8 mm to 2.2 mm, and for example, 2 mm.

液體流道306例如是具有深度的溝槽。在本實施例中，液體流道306具有液體交換區306A，其位於卵子培育與精卵結合系統200的第二貫孔部220下方。液體交換區306A例如是透過擴散而與第二貫孔部220進行液體交換，使得液體灌注系統300與卵子培育與精卵結合系統200進行液體交換。在本實施例中，液體交換區306A例如是具有與第二貫孔部220相符的輪廓，諸如圓形。The liquid flow path 306 is, for example, a groove having a depth. In this embodiment, the liquid flow channel 306 has a liquid exchange area 306A, which is located below the second through-hole portion 220 of the egg breeding and sperm-egg combination system 200. The liquid exchange area 306A performs liquid exchange with the second through-hole portion 220 by diffusion, for example, so that the liquid perfusion system 300 and the egg cultivation and sperm-egg combination system 200 perform liquid exchange. In the present embodiment, the liquid exchange area 306A has, for example, a contour conforming to the second through-hole portion 220, such as a circle.

在本實施例中，如圖1B所示，整合式晶片10例如是包括第一薄膜F1、位於第一薄膜F1下方的第二薄膜F2、位於第二薄膜F2下方的第三薄膜F3以及位於第三薄膜F3下方的第四薄膜F4。在本實施例中，第一薄膜F1至第四薄膜F4的材料例如是PDMS，但本發明不以此為限。在本實施例中，第一薄膜F1包括對應於***入口部102的第一通孔OP1、對應於***出口部104的第二通孔OP2、對應於主流道106與分支流道108的第一溝槽T1、阻擋結構110的突起圖案P、對應於卵子培育與精卵結合系統200的槽體202的第三通孔OP3、對應於液體入口部302的第四通孔OP4以及對應於液體出口部304的第五通孔OP5。其中，第一通孔OP1至第五通孔OP5例如是貫穿第一薄膜F1，以及第一溝槽T1例如是具有小於第一薄膜F1之厚度的深度。In this embodiment, as shown in FIG. 1B, the integrated wafer 10 includes, for example, a first film F1, a second film F2 located below the first film F1, a third film F3 located below the second film F2, and a The fourth film F4 under the three films F3. In this embodiment, the material of the first film F1 to the fourth film F4 is, for example, PDMS, but the invention is not limited thereto. In this embodiment, the first thin film F1 includes a first through hole OP1 corresponding to the sperm inlet 102, a second through hole OP2 corresponding to the sperm outlet 104, and a first corresponding to the main channel 106 and the branch channel 108 The groove T1, the protrusion pattern P of the blocking structure 110, the third through hole OP3 corresponding to the groove body 202 of the egg breeding and sperm and egg combining system 200, the fourth through hole OP4 corresponding to the liquid inlet portion 302, and the liquid outlet The fifth through hole OP5 of the portion 304. The first through hole OP1 to the fifth through hole OP5 are, for example, penetrating the first film F1, and the first trench T1 is, for example, having a depth smaller than the thickness of the first film F1.

第二薄膜F2包括第一貫孔部210、與第四通孔OP4對應的第六通孔OP6以及與第五通孔OP5對應的第七通孔OP7，其中位於第一通孔OP1、第二通孔OP2以及第一溝槽T1下方的第二薄膜F2分別作為***入口部102、***出口部104以及主流道106與分支流道108的底部。第三薄膜F3包括第二貫孔部220、與第六通孔OP6對應的第八通孔OP8以及與第七通孔OP7對應的第九通孔OP9。第四薄膜F4包括彼此相連的第一凹槽CV1、第二溝槽T2以及第二凹槽CV2，其中第一凹槽CV1對應於第八通孔OP8且作為液體入口部302的底部，第二溝槽T2作為液體流道，第二凹槽CV2對應於第九通孔OP9且作為液體出口部304的底部。換言之，第四通孔OP4、第六通孔OP6、第八通孔OP8以及第一凹槽CV1形成液體入口部302；第五通孔OP5、第七通孔OP7、第九通孔OP9以及第二凹槽CV2形成液體出口部304。The second film F2 includes a first through hole portion 210, a sixth through hole OP6 corresponding to the fourth through hole OP4, and a seventh through hole OP7 corresponding to the fifth through hole OP5, in which the first through hole OP1, the second The through hole OP2 and the second thin film F2 under the first trench T1 serve as the bottoms of the sperm inlet 102, the sperm outlet 104, and the main channel 106 and the branch channel 108, respectively. The third film F3 includes a second through hole portion 220, an eighth through hole OP8 corresponding to the sixth through hole OP6, and a ninth through hole OP9 corresponding to the seventh through hole OP7. The fourth film F4 includes a first groove CV1, a second groove T2, and a second groove CV2 connected to each other, wherein the first groove CV1 corresponds to the eighth through hole OP8 and serves as the bottom of the liquid inlet portion 302, the second The groove T2 serves as a liquid flow path, and the second groove CV2 corresponds to the ninth through hole OP9 and serves as the bottom of the liquid outlet portion 304. In other words, the fourth through hole OP4, the sixth through hole OP6, the eighth through hole OP8, and the first groove CV1 form the liquid inlet portion 302; the fifth through hole OP5, the seventh through hole OP7, the ninth through hole OP9, and the first The two grooves CV2 form a liquid outlet 304.

在本實施例中，例如是透過翻模以及鑽孔製程來製作整合式晶片10的第一薄膜F1至第四薄膜F4。以下將透過製作包括第一貫孔部的第二薄膜F2為例來進行說明，但可以理解的是，第一薄膜F1、第三薄膜F3以及第四薄膜F4亦可透過相似方法製作。首先，提供基板（未繪示），並以黃光微影製程於基板上形成作為母模的柱狀陣列結構（未繪示），其中柱狀陣列結構包括多個柱狀圖案，柱狀圖案的高度例如是第一貫孔的深度，以及柱狀圖案的直徑例如是第一貫孔的孔徑。基板例如是矽晶圓。柱狀陣列結構的材料例如是負光阻，諸如SU-8。接著，透過旋轉塗布等方式將PDMS等薄膜材料塗布至基板表面並填入柱狀陣列結構之間的間隙中，於固化薄膜材料之後，透過翻模得到包括第一貫孔部的薄膜。而後，以鑽孔製程來形成第六通孔OP6與第七通孔OP7。當然，除了上述方法以外，也可以使用諸如蝕刻等其他合適的方法來製作各膜層。In this embodiment, for example, the first film F1 to the fourth film F4 of the integrated wafer 10 are manufactured through a mold-turning and drilling process. The following description will be made by making the second film F2 including the first through-hole portion as an example, but it can be understood that the first film F1, the third film F3, and the fourth film F4 can also be made by a similar method. First, provide a substrate (not shown), and form a columnar array structure (not shown) as a master mold on the substrate using a yellow photolithography process, wherein the columnar array structure includes a plurality of columnar patterns, the height of the columnar patterns For example, the depth of the first through hole and the diameter of the columnar pattern are, for example, the diameter of the first through hole. The substrate is, for example, a silicon wafer. The material of the columnar array structure is, for example, negative photoresist, such as SU-8. Next, a thin film material such as PDMS is applied to the surface of the substrate by spin coating or the like and filled into the gaps between the columnar array structures. After the thin film material is cured, the thin film including the first through-hole portion is obtained by turning mold. Then, the sixth through hole OP6 and the seventh through hole OP7 are formed by a drilling process. Of course, in addition to the above methods, other suitable methods such as etching may also be used to make each film layer.

在本實施例中，例如是透過氧電漿改質方式來黏合第一薄膜F1至第四薄膜F4中的相鄰兩者。舉例來說，可分別對第二薄膜F2至第四薄膜F4的表面進行改質，並使其分別附著至第一薄膜F1至第三薄膜F3的底面，如此一來，使得第一薄膜F1至第四薄膜F4結合在一起，以形成整合式晶片10。在一實施例中，為了攜帶方便，可將整合式晶片10與一載板20結合，載板20的材料可為玻璃。再者，整合式晶片10可更包括密封件（未繪示），用以控制氣壓差異，使得液體暫時停止流動。舉例來說，密封件例如是覆蓋於***分選系統100上，以密封***分選系統100的***入口部102與***出口部104。再者，另一密封件例如是覆蓋於卵子培育與精卵結合系統200上，以密封槽體202。在一實施例中，密封件可以是諸如膠帶等具可密封性的物質。In this embodiment, for example, the adjacent two of the first film F1 to the fourth film F4 are bonded by oxygen plasma modification. For example, the surfaces of the second film F2 to the fourth film F4 can be modified and attached to the bottom surfaces of the first film F1 to the third film F3, respectively, so that the first film F1 to The fourth film F4 is bonded together to form the integrated wafer 10. In one embodiment, for convenience of carrying, the integrated chip 10 can be combined with a carrier 20, and the material of the carrier 20 can be glass. Furthermore, the integrated chip 10 may further include a seal (not shown) to control the difference in air pressure so that the liquid temporarily stops flowing. For example, the seal is covered on the sperm sorting system 100 to seal the sperm inlet 102 and sperm outlet 104 of the sperm sorting system 100, for example. Furthermore, another sealing element covers the egg cultivating and sperm-egg combination system 200 to seal the tank 202, for example. In an embodiment, the seal may be a sealable substance such as tape.

接下來將說明透過整合式晶片進行***分選、卵子培育以及體外受精的方法。Next, methods of sperm sorting, egg cultivation, and in vitro fertilization through integrated chips will be explained.

首先，提供整合式晶片10。接著，對***分選系統100進行潤濕步驟，以潤濕主流道106與分支流道108。具體來說，將潤濕液體滴入***入口部102，使得液體流經主流道106與分支流道108，而使其潤濕。在本實施例中，用於潤濕步驟的液體例如是生理食鹽水、生殖細胞培養液或其他與***具有等張壓力的液體。First, the integrated chip 10 is provided. Next, a wetting step is performed on the sperm sorting system 100 to wet the main channel 106 and the branch channel 108. Specifically, the wetting liquid is dropped into the sperm inlet 102 so that the liquid flows through the main flow path 106 and the branch flow path 108 to wet it. In the present embodiment, the liquid used for the wetting step is, for example, physiological saline, germ cell culture liquid, or other liquid having isotonic pressure with sperm.

接著，將含卵子的樣本液滴入卵子培育與精卵結合系統200的槽體202中，使得卵子分別位於第一貫孔212中。在本實施例中，透過控制第一貫孔212的尺寸，一個第一貫孔212中例如是僅配置有一顆卵子。然後，對卵子進行前處理步驟。在本實施例中，前處理步驟例如是卵子透明帶去除步驟，此步驟例如是透過於槽體202中添加酵素來進行。在本實施例中，於完成前處理步驟之後（例如酵素作用完全之後），更包括開啟液體灌注系統300。也就是說，將液體B供給至液體入口部302，使得液體B流經液體流道306而進入液體出口部304。液體B可以是用於與槽體202中的液體進行交換的液體，諸如生理食鹽水、生殖細胞培養液或其他與細胞具有等張壓力的液體。在本實施例中，由於第二貫孔部220與其下方的液體流道306（即液體交換區306A）之間存在壓力差異，因此槽體202中的諸如酵素等有害物質可通過第二貫孔部220進入液體流道306而被排出。此外，液體流道306中的液體B亦可透過第二貫孔部220進入槽體202中，以進行液體交換。如此一來，能使卵子培育與精卵結合系統200的槽體202維持適合培育卵子或受精卵的環境。而後，可透過密封件暫時將槽體202密封。此處，槽體202的密封能阻擋分支流道108與槽體202之間的連通，以避免物質（諸如非所要的***）於此時由分支流道108進入槽體202。Next, the egg-containing sample is dropped into the tank 202 of the egg culturing and sperm-egg combination system 200 so that the eggs are located in the first through holes 212, respectively. In this embodiment, by controlling the size of the first through hole 212, for example, only one egg is disposed in one first through hole 212. Then, the egg is subjected to pretreatment steps. In this embodiment, the pretreatment step is, for example, a zona pellucida removal step. This step is performed by adding an enzyme to the tank 202, for example. In this embodiment, after completing the pre-treatment step (for example, after the enzyme has been completely activated), it further includes turning on the liquid perfusion system 300. That is, the liquid B is supplied to the liquid inlet 302 so that the liquid B flows through the liquid flow path 306 and enters the liquid outlet 304. The liquid B may be a liquid used for exchange with the liquid in the tank 202, such as physiological saline, germ cell culture liquid, or other liquid having isotonic pressure with the cells. In this embodiment, since there is a pressure difference between the second through-hole portion 220 and the liquid flow path 306 (ie, the liquid exchange area 306A) underneath, harmful substances such as enzymes in the tank 202 can pass through the second through-hole The part 220 enters the liquid flow path 306 and is discharged. In addition, the liquid B in the liquid flow path 306 can also enter the tank 202 through the second through-hole portion 220 for liquid exchange. In this way, the tank 202 of the egg cultivating and sperm-egg combining system 200 can maintain an environment suitable for cultivating eggs or fertilized eggs. Then, the tank 202 can be temporarily sealed through the seal. Here, the seal of the tank 202 can block the communication between the branch flow channel 108 and the tank body 202 to prevent substances (such as undesired sperm) from entering the tank body 202 from the branch flow channel 108 at this time.

接著，將含***的樣本液滴入***入口部102，使得***分選系統100對含***的樣本液進行***分選。在本實施例中，樣本液可為人類、豬、牛、馬等具有***的***。然後，於完成***分選步驟之後，將卵子培育與精卵結合系統200打開，使得分選後的高效能***通過分支流道108進入槽體202，以與卵子結合成受精卵。此處，分選後的高效能***為能保持在分支流道108附近的***，其例如是具有中等泳動力的***。在本實施例中，分選時間例如是10分鐘左右，此時的活***所佔的比例約85%左右。在本實施例中，打開卵子培育與精卵結合系統200的方法例如是移除密封件。此外，在受精卵形成後，可以進一步原位培養受精卵。原位培養受精卵的過程包括更換槽體202中的培養液，具體來說，可以透過抽乾槽體202中的培養液，再補充新鮮的培養液來進行。此外，一般來說，由於含***的樣本液中通常還含有雜質，因此，在一實施例中，可以進一步在***入口部102中設置過濾構件，使得含***的樣本液在進入主流道106進行分選之前先進行過濾，以避免雜質影響後續分選等過程。在一實施例中，過濾構件可以是相似於第二貫孔部220的過濾膜，舉例來說，過濾膜的材料可以是PDMS，且過濾膜的貫孔的孔徑與深度例如分別是25μm至35μm與50μm至100μm。Next, the sperm-containing sample is dropped into the sperm inlet 102, so that the sperm sorting system 100 performs sperm sorting on the sperm-containing sample liquid. In this embodiment, the sample fluid may be sperm with sperm such as human, pig, cow, horse, etc. Then, after completing the sperm sorting step, the egg cultivation and sperm and egg combination system 200 is opened, so that the sorted high-performance sperm enters the trough 202 through the branch flow channel 108 to combine with the egg to form a fertilized egg. Here, the sorted high-performance sperm is sperm that can be kept near the branch flow channel 108, which is, for example, sperm with moderate swimming power. In this embodiment, the sorting time is, for example, about 10 minutes, and the proportion of live sperm at this time is about 85%. In this embodiment, the method of opening the egg cultivating and sperm-egg combining system 200 is, for example, to remove the seal. In addition, after the fertilized egg is formed, the fertilized egg can be further cultured in situ. The process of cultivating the fertilized egg in situ includes replacing the culture solution in the tank 202. Specifically, it can be performed by draining the culture solution in the tank 202 and replenishing the fresh culture solution. In addition, generally speaking, since the sperm-containing sample liquid usually also contains impurities, in one embodiment, a filter member may be further provided in the sperm inlet portion 102 so that the sperm-containing sample liquid enters the main flow channel 106 Filter before sorting to avoid impurities affecting subsequent sorting and other processes. In an embodiment, the filter member may be a filter membrane similar to the second through-hole portion 220, for example, the material of the filter membrane may be PDMS, and the pore size and depth of the through-hole of the filter membrane are, for example, 25 μm to 35 μm, respectively With 50μm to 100μm.

綜上所述，本發明將***分選系統、卵子培育與精卵結合系統以及液體灌注系統整合在單一晶片上，使得經***分選系統分選後的高效能***能直接被導入卵子培育與精卵結合系統，以與經培育的卵子原位結合成受精卵，進而達成體外受精的目的。特別是，在本發明中，僅需將包括***的樣本液滴入***入口部，以及將包括卵子的樣本液滴入卵子培育與精卵結合系統的槽體，而無須進行其他主動式處理，就能被動式地分選***、處理卵子以及形成受精卵，故過程溫和而能避免傷害精卵，使得精卵存活率與結合率大幅提升。因此，本發明的晶片不僅能縮短傳統人工生殖技術之操作時間，更透過簡化操作流程來避免因操作時間冗長、人為操作失當而導致的檢體死亡，進而大幅地提升人工生殖技術的成功率。In summary, the present invention integrates the sperm sorting system, egg cultivation and sperm-egg combination system, and liquid perfusion system on a single chip, so that the high-performance sperm sorted by the sperm sorting system can be directly introduced into the egg cultivation and The sperm-egg binding system combines with the cultivated egg in situ to form a fertilized egg, thereby achieving the purpose of in vitro fertilization. In particular, in the present invention, it is only necessary to drop a sample including sperm into the sperm inlet, and drop a sample including egg into the tank of the egg cultivating and sperm-egg system, without performing other active treatments, It can passively sort sperm, process eggs and form fertilized eggs, so the process is gentle and can avoid sperm eggs damage, so that the survival rate and combination rate of sperm eggs are greatly improved. Therefore, the chip of the present invention can not only shorten the operation time of the traditional artificial reproduction technology, but also avoid the death of the specimen caused by the lengthy operation time and the artificial improper operation by simplifying the operation process, thereby greatly improving the success rate of the artificial reproduction technology.

雖然本發明已以實施例揭露如上，然其並非用以限定本發明，任何所屬技術領域中具有通常知識者，在不脫離本發明的精神和範圍內，當可作些許的更動與潤飾，故本發明的保護範圍當視後附的申請專利範圍所界定者為準。Although the present invention has been disclosed as above with examples, it is not intended to limit the present invention. Any person with ordinary knowledge in the technical field can make some changes and modifications without departing from the spirit and scope of the present invention. The scope of protection of the present invention shall be subject to the scope defined in the appended patent application.

10:整合式晶片20:載板100:***分選系統102:***入口部104:***出口部106:主流道106A、106B、106C:區段108:分支流道110:阻擋結構110A:頭端110B:連接端110C:末端110D:集精槽200:卵子培育與精卵結合系統202:槽體210:第一貫孔部212:第一貫孔220:第二貫孔部222:第二貫孔300:液體灌注系統302:液體入口部304:液體出口部306:液體流道306A:液體交換區d:距離10: Integrated chip 20: Carrier board 100: Sperm sorting system 102: Sperm inlet 104: Sperm outlet 106: Main channel 106A, 106B, 106C: Section 108: Branch channel 110: Barrier structure 110A: Head end 110B: connecting end 110C: end 110D: sperm collecting tank 200: egg breeding and sperm and egg combining system 202: tank body 210: first through hole portion 212: first through hole 220: second through hole portion 222: second through hole Hole 300: liquid perfusion system 302: liquid inlet 304: liquid outlet 306: liquid flow path 306A: liquid exchange area d: distance

圖1A是依照本發明的實施例的一種整合式晶片的立體示意圖，圖1B是圖1A的整合式晶片的***示意圖，圖1C是圖1A的整合式晶片的***分選系統的仰視圖，以及圖1D是圖1A的整合式晶片的剖面示意圖。1A is a schematic perspective view of an integrated chip according to an embodiment of the present invention, FIG. 1B is an exploded schematic view of the integrated chip of FIG. 1A, FIG. 1C is a bottom view of the sperm sorting system of the integrated chip of FIG. 1A, and FIG. 1D is a schematic cross-sectional view of the integrated chip of FIG. 1A.

10:整合式晶片 10: Integrated chip

20:載板 20: Carrier board

100:***分選系統 100: Sperm sorting system

102:***入口部 102: Sperm entrance

104:***出口部 104: Department of Sperm Export

106:主流道 106: Mainstream

108:分支流道 108: branch channel

110:阻擋結構 110: blocking structure

200:卵子培育與精卵結合系統 200: Egg cultivation and sperm and egg combination system

202:槽體 202: tank body

300:液體灌注系統 300: Liquid perfusion system

302:液體入口部 302: Liquid inlet

304:液體出口部 304: Liquid outlet

306:液體流道 306: Liquid flow path

Claims

一種用於***分選、卵子培育以及體外受精的整合式晶片，包括： ***分選系統，包括***入口部、***出口部、主流道以及分支流道，所述主流道與所述***入口部及所述***出口部連通，所述主流道具有由所述***入口部朝向所述***出口部逐漸增大的寬度，所述分支流道位於所述***入口部與所述***出口部之間且與所述主流道連通；卵子培育與精卵結合系統，與所述分支流道連通，包括第一貫孔部與第二貫孔部，其中所述第一貫孔部包括多個第一貫孔，所述第一貫孔的孔徑大於卵子的平均直徑，用以容納待與***結合的卵子，所述第二貫孔部位於所述第一貫孔部下方且包括多個第二貫孔，所述第二貫孔的孔徑小於卵子，用以進行液體交換，其中經所述***分選系統分選後的***透過所述分支流道被導入所述卵子培育與精卵結合系統，以與位於所述第一貫孔中的卵子結合；以及液體灌注系統，包括液體入口部、液體出口部以及液體流道，其中所述液體流道位於所述液體入口部及所述液體出口部之間且與所述液體入口部及所述液體出口部連通，所述液體流道的一部分位於所述第二貫孔部下方且通過所述第二貫孔部與所述卵子培育與精卵結合系統進行液體交換。An integrated chip for sperm sorting, egg cultivation, and in vitro fertilization, including: a sperm sorting system, including a sperm inlet, a sperm outlet, a mainstream channel, and a branch channel, the mainstream channel and the sperm inlet Communicating with the sperm outlet, the main flow channel has a width that gradually increases from the sperm inlet to the sperm outlet, and the branch flow channel is located between the sperm inlet and the sperm outlet And communicates with the main flow channel; an egg breeding and sperm-egg combination system, which communicates with the branch flow channel, includes a first through hole portion and a second through hole portion, wherein the first through hole portion includes a plurality of first A through hole, the diameter of the first through hole is larger than the average diameter of the egg for accommodating the egg to be combined with sperm, the second through hole portion is located below the first through hole portion and includes a plurality of second through holes Hole, the diameter of the second through hole is smaller than the egg, for liquid exchange, wherein the sperm sorted by the sperm sorting system is introduced into the egg cultivating and sperm-egg system through the branch flow channel, To combine with the egg located in the first through hole; and a liquid perfusion system, including a liquid inlet, a liquid outlet, and a liquid flow channel, wherein the liquid flow channel is located at the liquid inlet and the liquid outlet Between and in communication with the liquid inlet portion and the liquid outlet portion, a portion of the liquid flow path is located below the second through-hole portion and passes through the second through-hole portion with the egg breeding and sperm eggs Liquid exchange in combination with the system.

如申請專利範圍第1項所述的整合式晶片，其中所述第一貫孔部與所述第二貫孔部的材料包括聚二甲基矽氧烷。The integrated chip as described in item 1 of the patent application range, wherein the materials of the first through-hole portion and the second through-hole portion include polydimethylsiloxane.

如申請專利範圍第1項所述的整合式晶片，其中所述第一貫孔是規則排列。The integrated chip as described in item 1 of the patent application scope, wherein the first through holes are regularly arranged.

如申請專利範圍第1項所述的整合式晶片，其中所述第二貫孔是規則排列。The integrated chip as described in item 1 of the patent application scope, wherein the second through holes are regularly arranged.

如申請專利範圍第1項所述的整合式晶片，其中所述第二貫孔的孔徑大於30um。The integrated chip as described in item 1 of the patent application scope, wherein the diameter of the second through hole is greater than 30 um.

如申請專利範圍第1項所述的整合式晶片，其中所述主流道包括第一區段及與所述第一區段連接的第二區段，所述第一區段位於所述***入口部與所述第二區段之間，所述第二區段位於所述第一區段與所述***出口部之間，其中所述第一區段具有實質上相同的寬度，所述第二區段具有逐漸增大的寬度且其最小寬度大於所述第一區段的所述寬度。The integrated chip according to item 1 of the patent application scope, wherein the main flow path includes a first section and a second section connected to the first section, the first section is located at the sperm inlet Between the first section and the second section, the second section is located between the first section and the sperm outlet section, wherein the first section has substantially the same width, the first section The second section has a gradually increasing width and its minimum width is greater than the width of the first section.

如申請專利範圍第6項所述的整合式晶片，其中所述分支流道的寬度小於所述第二區段的寬度。The integrated wafer according to item 6 of the patent application range, wherein the width of the branch flow channel is smaller than the width of the second section.

如申請專利範圍第1項所述的整合式晶片，其中所述***分選系統更包括阻擋結構，所述阻擋結構位於所述主流道中且鄰近於所述***出口部，且所述阻擋結構與所述主流道的側壁之間具有距離。The integrated chip as described in item 1 of the patent application scope, wherein the sperm sorting system further includes a blocking structure, the blocking structure is located in the main channel and is adjacent to the sperm outlet, and the blocking structure is There is a distance between the side walls of the main channel.

如申請專利範圍第8項所述的整合式晶片，其中所述阻擋結構為啞鈴形。The integrated chip as described in item 8 of the patent application range, wherein the barrier structure is dumbbell-shaped.

如申請專利範圍第1項所述的整合式晶片，其中所述***入口部與所述***出口部為槽體。The integrated wafer according to item 1 of the patent application scope, wherein the sperm inlet and the sperm outlet are grooves.

如申請專利範圍第1項所述的整合式晶片，其中所述液體流道具有液體交換區，位於所述卵子培育與精卵結合系統下方且具有與所述第二貫孔部相符的輪廓。The integrated wafer as described in item 1 of the patent application range, wherein the liquid flow channel has a liquid exchange area, is located below the egg cultivating and sperm-egg combination system, and has a contour conforming to the second through-hole portion.

如申請專利範圍第1項所述的整合式晶片，包括：第一薄膜，包括對應於所述***入口部的第一通孔、對應於所述***出口部的第二通孔、對應於所述主流道與所述分支流道的第一溝槽、對應於所述卵子培育與精卵結合系統的第三通孔、對應於所述液體入口部的第四通孔以及對應於所述液體出口部的第五通孔；位於所述第一薄膜下方的第二薄膜，包括所述第一貫孔部、與所述第四通孔對應的第六通孔以及與所述第五通孔對應的第七通孔，其中位於所述第一通孔、所述第二通孔以及所述第一溝槽下方的所述第二薄膜分別作為所述***入口部、所述***出口部以及所述主流道與所述分支流道的底部；位於所述第二薄膜下方的第三薄膜，包括所述第二貫孔部、與所述第六通孔對應的第八通孔以及與所述第七通孔對應的第九通孔；以及位於所述第三薄膜下方的第四薄膜，包括彼此相連的第一凹槽、第二溝槽以及第二凹槽，其中所述第一凹槽對應於所述第八通孔且作為所述液體入口部的底部，所述第二溝槽作為所述液體流道，所述第二凹槽對應於所述第九通孔且作為所述液體出口部的底部。The integrated chip as described in item 1 of the patent application scope includes: a first thin film, including a first through hole corresponding to the sperm inlet, a second through hole corresponding to the sperm outlet, corresponding to the A first groove of the main flow path and the branch flow path, a third through hole corresponding to the egg cultivating and sperm-egg combination system, a fourth through hole corresponding to the liquid inlet portion, and corresponding to the liquid A fifth through hole in the exit portion; a second film located below the first film, including the first through hole portion, the sixth through hole corresponding to the fourth through hole, and the fifth through hole A corresponding seventh through hole, wherein the second thin film located below the first through hole, the second through hole, and the first trench respectively serves as the sperm inlet, the sperm outlet, and The main flow channel and the bottom of the branch flow channel; a third film located below the second film, including the second through hole portion, the eighth through hole corresponding to the sixth through hole, and the A ninth through hole corresponding to the seventh through hole; and a fourth film under the third film, including a first groove, a second groove and a second groove connected to each other, wherein the first groove The groove corresponds to the eighth through hole and serves as the bottom of the liquid inlet portion, the second groove serves as the liquid flow path, and the second groove corresponds to the ninth through hole and serves as the The bottom of the liquid outlet.

如申請專利範圍第12項所述的整合式晶片，其中所述第一薄膜、所述第二薄膜、所述第三薄膜以及所述第四薄膜的材料包括聚二甲基矽氧烷。The integrated wafer as described in item 12 of the patent application range, wherein the material of the first film, the second film, the third film and the fourth film includes polydimethylsiloxane.

如申請專利範圍第13項所述的整合式晶片，其中所述第一薄膜、所述第二薄膜、所述第三薄膜以及所述第四薄膜中相鄰兩者彼此黏合。The integrated wafer according to item 13 of the patent application range, wherein adjacent ones of the first film, the second film, the third film, and the fourth film are bonded to each other.

一種用於***分選、卵子培育以及體外受精的方法，包括：提供如申請專利範圍第1項所述的整合式晶片；將含***的樣本液滴入所述***入口部中，使得所述***分選系統對所述含***的樣本液進行***分選；將含卵子的樣本液滴入所述卵子培育與精卵結合系統中，使得所述卵子分別位於所述第一貫孔中；以及使經分選的所述***通過所述分支流道被導入所述卵子培育與精卵結合系統，以於所述第一貫孔中與所述卵子結合成受精卵。A method for sperm sorting, egg cultivation, and in vitro fertilization, including: providing an integrated chip as described in item 1 of the patent application scope; dropping a sperm-containing sample into the sperm inlet so that the The sperm sorting system performs sperm sorting on the sperm-containing sample solution; dropping the egg-containing sample into the egg cultivating and sperm-egg combining system so that the eggs are located in the first through holes respectively; And the sorted sperm is introduced into the egg cultivating and sperm egg combining system through the branch flow channel to combine with the egg in the first through hole to form a fertilized egg.

如申請專利範圍第15項所述的方法，更包括在將所述含卵子的樣本液滴入所述卵子培育與精卵結合系統中之後，對所述卵子進行透明帶去除步驟。The method as described in item 15 of the scope of the patent application further includes the step of removing the zona pellucida after dropping the egg-containing sample into the egg breeding and sperm-egg combination system.

如申請專利範圍第15項所述的方法，更包括在將所述含卵子的樣本液滴入所述卵子培育與精卵結合系統中之後，密封所述卵子培育與精卵結合系統，以及在所述***分選系統對所述含***的樣本液完成所述***分選之後，通過打開所述卵子培育與精卵結合系統使得經分選的所述***通過所述分支流道進入所述卵子培育與精卵結合系統。The method according to item 15 of the scope of the patent application further includes sealing the egg-cultivating and sperm-egg combining system after dropping the egg-containing sample into the egg-cultivating and sperm-egg combining system, and After the sperm sorting system completes the sperm sorting on the sperm-containing sample liquid, the sperm sorted sperm enters the branch flow channel by opening the egg cultivation and sperm egg combination system Egg breeding and sperm and egg combination system.

如申請專利範圍第15項所述的方法，更包括在將所述含***的樣本液滴入所述***入口部中之後，密封所述***分選系統，以及在將含卵子的樣本液滴入所述卵子培育與精卵結合系統中之後，通過打開所述***分選系統以進行所述***分選。The method according to item 15 of the scope of the patent application further includes sealing the sperm sorting system after dropping the sperm-containing sample droplet into the sperm inlet, and applying the egg-containing sample droplet After entering the egg cultivating and sperm-egg combining system, the sperm sorting is performed by turning on the sperm sorting system.

如申請專利範圍第15項所述的方法，更包括在所述卵子培育與精卵結合系統中培養所述受精卵。The method as described in item 15 of the patent application scope further includes cultivating the fertilized egg in the egg cultivating and sperm egg combination system.

如申請專利範圍第15項所述的方法，更包括使液體在所述液體灌注系統中流動，其中所述液體經通過所述第二貫孔部與所述卵子培育與精卵結合系統中的液體進行液體交換。The method as described in item 15 of the patent application scope, further comprising flowing a liquid in the liquid perfusion system, wherein the liquid passes through the second through-hole portion and the egg cultivation and sperm-egg combination system Liquid exchanges.