200538735 玖、發明說明： 【發明所屬之技術領域】 本發明是有關於一種細胞計數分類晶片及其製造方法 ’特別是指一種利用邊鞘流聚焦樣品流體寬度且以蝕刻技 5 術製作的細胞計數分類晶片。 【先前技術】 一般於生物科技業及學界中之細胞計數方式大多是使 用如圖1所示的由Miyaky所提出之細胞計數器}以邊鞘流 聚焦一樣品流體的方式，將該樣品流體聚焦至寬度僅容一 _ 10 15 20 細胞流過的寬度，再計數該樣品流體中之細胞數量。該細 见计數B曰片中形成一邊鞘流就須接引一管路丨丨〇至該細胞計 數器，再加上注入樣品流體之管路U1及輸出流體之管路 112就需四條管路。因此，如須同時偵測多種樣品流體時， 須接引更多之管線以形成更多之邊鞠流，於測試及組裝上 都不方便。 參閱圖2，由於該㈣計數器1是以複數财不同形狀 之簍空槽孔的玻璃丨12〜16互相疊貼以形成複數中空之流 體通道，並注人樣品流體及水進人該等料，使水形成邊 勒流夾擠聚焦該樣品流體，容㈣成玻魏i2〜i6間縫隙 漏洩流體，且於製作上也較為繁雜。 另外細胞叶數之方法大略有兩種，第-種是利用榮 光標定來檢測經染色做為標籤的細胞，以雷射隼中光束於 射出來的光，即=、,=射雷射光後放射或是散 道細胞的大小、形狀及化學組成成份 6 200538735 :在實際的應用時，細胞必須準確地經過量測區域的雷射 光束正中a，以確保能得到最大的輸出訊號。但上述之細 胞計數方式需先將細胞染色，製作費時又麻煩，且其使用 設備需要極高精準度的流體動力及雷射光束，因此應用此 =式所組合的細胞計數器及儀器設備體積都很龐大且價格 昂貴，使用成本較高。 ίο 15 ^另一種細胞計數器是在細胞流體聚焦的一管道末端埋 藏入一對軸向對應於該管道兩側的光纖並通入雷射光，利 用細胞經過光纖時，細胞_二光纖間之光線，引起光線鲁 的變化來谓測到細胞。而其流體驅動是以整合電極來驅動 細胞流聚焦’使細胞皆維持同一高度，細胞引起之光線訊 號變化強度-致。但此項技術需製作電極，光纖對準等精 密之程序，製程較為繁雜。 【發明内容】 於是，本發明即在提供-種製作簡易、不易茂漏及低 製作成本具可同時❹I多條細胞流並予以分類的細胞計數 分類晶片及其製作方法。本發明另一目的即在提供一種使 用成本低廉，不須昂貴設備儀器的細胞檢測方法。 本發明細胞計數分類晶片，適用於計數一樣品流體内 之細胞數量，該細胞計數分類晶片包含：一透明基座，及 一中空地形成於該基座内的微管道單元。該微管道單元具 有：一主通道、二互相平行間隔排列於該主通道之二側的 副通道，及一連通於該主通道與該等副通道之末端的匯流 通道。該主通道之頭端連通至一形成於該基座表面供所述 20 200538735 樣品流體進入之導入孔。該等副通道之頭端互相連通並連 通至一形成於該基座表面供水流體進入之導入孔。該匯流 通道是用於匯流接引樣品流體及水流體並連通至一形成於 該基座表面之流出孔。該二副通道中之水流體流至該匯流 通道時形成二邊鞘流，該二邊鞠流限制由主管道流出之樣 品流體的寬度至單一細胞可流通之寬度，並由監測流經之 細胞數量計算樣品流體内含之細胞數量。 該細胞計數分類晶片之製造方法包含以下步驟： ίο 15 (A)於一水平之基板之頂面上蝕刻出一主通道、一平_ 行間隔於該主通道兩側的副通道，及頭端共同連通主通道 及該等副通道末端的一匯流通道，該等副通道之頭端是互 相連通。（B )在該基板之該等主通道之頭端以鑽孔機鑽出 貝穿至该基板底面之導入孔，於該等副通道之頭端共同 連通處鑽出一貫穿至該基板底面的注入孔，及於該匯流通 道之末端鑽出一貫穿至該基板底面的流出孔。（c)把一玻 璃上板覆蓋於該基板頂面並進行高溫接合。 而使用《亥細胞汁數分類晶片檢測細胞之方法係使用一 數位攝#機_取影像的方式檢測該細胞計數分類晶片内之 樣品流體的細胞，所述樣品流體之寬度僅容一細胞單獨流 、/、田I檢/則方法之步驟如下··（ A )以所述數位攝影機 :攝所述樣品流體㈣之數位影像。（b)圈選該數位影像 中樣品流體之流程中的一 、 ^又洛作為"一第' 一區域。（C )臣七 測該第一區域中之與俏七μ /皿 以中之衫像灰階值。（D)計數該第一區域 像灰階值改變次數，兮势 〜 Μ第一區域中影像灰階值改變之次數 20 200538735 就代表細胞流過之數目。 本發明之功效能在於提供一設備構造簡單，並節省封 裝、測試時間，簡化製作程序，且減低縫隙线漏之機會。1 並可偵測細胞數目及種類，且可藉著施加介電泳力於:管 5 道單元末端進行細胞分類回收的細胞計數分類晶片及其 造與使用方法。 ^ 【實施方式】 有關本發明之前述及其他技術内容、特點與功效，在 以下配合參考圖式之一較佳實施例的詳細說明中， 10 楚的明白。 Θ 參閱圖3、圖4與圖5，該細胞計數分類晶片是用於計 數㈣樣品流體2内之細胞數量，該細胞計數分類晶片包 括透明之基座3、-中空地形成於該基座3内的微管道單 元4，及一插伸於該微管道單元4内之電極單元7。 15 '亥基座3是由透明之鈉玻璃材質製作而成，概略成水 平之板狀。 5亥试官道單元4具有：二水平中空的主通道41、四互 相平行間隔排列於該主通道41旁的副通道42、一接續於該 2〇 Λ 田J通道41、42末端並管徑寬度漸縮的頸通道43、- 接續連通該等副通道42之頭端的輸水通道Μ、一連通於該 、込43末舳的匯流通道45，及連通於該匯流通道45頭 潢末端間並分別向兩側延伸至該基座3外的一第一分類通 、卜6與第一分類通道47。該四副通道42是兩兩成對， 卜對通道42是分別平行排列於一主通道41之兩側。 9 200538735 該等主通道41於連通該頸通道43虚 縮的主噴嘴411，㈣… 成—通道寬度漸 幵》成一、畜 以田、I 42於連通該頸通道43處亦各 瓜成:通道寬度漸縮的副嗔嘴421。該頸通道43 j度;I，41、42處寬度較寬’接通該匯流通道45處之 見度較乍，概略形成一漏斗形狀。於本實施例中是 通道W及四副通道42以分析兩種樣品流體2内 =’貫際上—主通道41配合二副通道C就可分析—種 ，需同時分析多種樣品流體2時，就增加主通道“及 ίο 15 20 副通道42之數量即可應用，因此實施上不以上述之數 限。 …、 該等主通道41之頭端内周面上分別形成一貫穿至該基 座3之底面的導人孔412，以分別供所述兩種樣品流體2流 入該二主通道41内。該輸水通道44頭端内周面上形成一 貫穿至該基座3底面的一注入孔441，並分支出複數分歧段 442，該等分歧段442就是連通至該等副通道42之頭端， 亦即該四副通道42之頭端是藉由該輸水通道44來共同連 通，也藉由該輸水通道44以導引水流體進入該四副通道42 _ 。本實施例中水流體是去離子水，但只要水中無明顯雜質 ，亦可以用普通水做為水流體。該匯流通道45之末端之内 周面上形成一貫穿至該基座3底面以將樣品流體2及水流 體排流出基座3外的流出孔451。該第一、第二分類通道 46、47是分別由該匯流通道45岔流分支出來，並分別延伸 至形成於3亥基座3底面的一第一分類孔461與一第二分類 孔 471。 10 ίο 15 20 200538735 一该電極單元7具有-插伸於難流通道45内鄰近該第 :、二分類通道46、47連通處的第一電極η、一插伸於該 :一分類通道46内並往第-電極延伸的第二電極72、一插 料該匯流通道45内並往第—電極延伸的第三電極乃，及 :插伸於該第二分類通道47内並往第—電極延伸的第四電 =4。藉此，施加電祕第—與第二電極7卜72間或第一 1 I71'73.或第—與第四電極間，以 於该Μ管道單元4末端之流矽甚 ν /瓜體產生介電泳力驅動不同之細 2別流人《流通道45末端或該第—、第二分類通道461 7内。本貫施例甲是以二分類通道作說明，實施上以— ^刀類通道就可達成分類之功效。 參閱圖4及圖6，本發明身丨丨田八 月利用分流之觀念1 一個泵浦 推動水流體進入注入孔44丨，經 副通道42,以喷流四條均勾邊:二“至四 法放 Ί邊勒机422經頸通道43進入匯 机目道。使用此分流原理’以此類推，即可在同一平面以 :=、51推動，以獲得所需的邊鞠流422數目。不 :了 51數里之使用’也解省封裝及測試之時間，更· 5\作1動之水流體流速平穩。本實施例中是以螺動式泵浦 為晶片内流體之驅動力’實施上不以此為限。 藉此’该—主通道41 、、φ > 丁彳彳 /主入兩種不同之樣品流體2，而 该專副通道42是由該泵浦 而 由該主、副通道41、42之主水桃體虽所有流體經 入該頸通道43,並匯流進入^嘴嘴411、421後’匯集嗔 立匯机進入该匯流通道45中時，每_主 之兩側副通道42中流出之水流體形成二邊勒流422, 11 200538735 該二邊鞘流422對併夾於其中央描σ 、τ天之樣卩卩流體2進行擠壓， 形成流體聚焦效應，此時適當妯胡敕、真灿^ 才過田地凋整邊鞘流422與樣品流 體2之流速比可將樣品流體2之甯$ ^ 匕耻ζ <羌度％減至數微米寬，也 就是該二邊鞘流422可限制樣品流體2之寬度至僅容單一 細胞通過的寬度，運用顯微鏡（圖未示）就二數：述樣 品流體2中細胞之數量。上述之水流體是去離子水，但只 要水中無明顯雜質’亦可以用普通水做為水流體。但隨著 水流體組成液體種類不同，流體ψ隹 4 ’爪般♦焦的所需的速度比亦會 不一樣。 ίο 15 20 此外，經過邊勒流422擠壓之樣品流冑2中經過計數 之後，可藉由施加電壓於該電極單元7之不同電極之間， 以驅動樣品流體中之不同細胞分別由不同之通道輸出至該 基座3外。例如，經計數觀察後之細胞如須分類收集至第 -分類孔461時’可施加一交流電壓於該第一電極7ι與第 電木72間以產生一正介電泳力以吸引細胞經由該第一 分類通道46往第一分類孔461輸出至該基座3外收集。運 用相同之原理就可職不同種類之細胞分別經由該等分類 孔461、471或流出孔451輸出至該基座3外以方便回收再 利用。於上例說明中，亦可用施加電麗產生負介電泳力以 推斥細胞由其他通道輸出的方式來分類細胞，實施上不以 上述之方法為限。另外該等分類通道及電極單元7之主要 目的是為分類細胞而設置，於實施上亦可不設置分類通道 與電極單元，仍然具有細胞計數之功能，因此實施範圍不 以此為限。 12 200538735 於本實施例之晶片中各通道之詳細尺寸請參考圖4，其 中該主喷嘴411之寬度為100微米，該副通道42之寬度為 150微米，而匯流通道45之寬度為.24〇微米，而圓6為調 整流速比後，於該匯流通道45中該樣品流體2可聚焦至寬 5 度達17微米之顯微照片。 參閱圖7,以下續對本發明細胞計數分類晶片之製造方 法加以說明： (A) 於一水平之玻璃基板31之頂面上塗佈上一光阻 劑層33，再以繪製有如圖4之微通道系統4之圖案的光罩籲 10 34覆蓋於該光阻劑層33上.，以顯影之技術將該通道分布轉 移至該基板31上，再以玻璃蝕刻液蝕刻該微管道單元 該微管道單元已於前詳述，不再贅述。 (B) 在該基板31之該等主通道41之頭端端以鑽孔機 鑽出-貫穿至該基板31底面之導入孔412，於該輸水通道 15 44之頭端鑽出一貫穿至該基板31底面的注入孔々μ、於該 匯流通道45之末端一鑽出一貫穿至該基板31底面的流出 孔45卜及分別於該第一、第二分類通道46、〇末端鑽出φ 一貫穿至該基板31底面的一第一分類孔461與第二分類孔 ' 471。再使用硫酸與過氧化氳的混和溶液以煮沸十分鐘的方· 20 式將該基板清洗後再烘乾。 (C) 於玻璃上板32之底面上以蒸鍍的方式先後舖 設-層鉻（CO金屬及一層金（Au)金屬，再以顯影蝕刻 的方式將該電極單元7以外之金屬刻除，並以丙酮、異丙 醇（Is0-pr0pyl Alcohol，IPA )、去離子水循序沖洗並烘乾。 13 200538735 因為巫與玻璃黏著性不好，所以利用絡金屬當中間的介面 使金金屬層可良好附著於該上板底面。 (D)把4上板32定位覆蓋於該基板^頂面並於一充 滿氮氣環境之高溫燒結爐中，利用高溫加熱上板Μ及基板 31使玻璃呈現雜狀態而接合，以使該等通道中空地形成 於該基板31及上板32間。在氮氣環境下進行高溫接合可 避免金屬電極發生氧化而造成晶片損毀。 (Ε)將接合好之上板32及基板31進行封裝，即形成 一細胞計數分類晶片。 ίο 以本製作方法製作出細胞計數分類晶片具有以下之優 點： 32 15 〇)僅以一蝕刻該等通道後之玻璃基板31及一上板 、相互疊接独高溫接合，比習知五玻躲㈣合之結構 減少玻璃板間之隙_漏之機會，也簡化製作程序。 ⑺本製作方法可製作出可同時檢測多種樣品流體2 之曰曰片’只須㈣影程序時使用緣製不同通道結構之光罩 34 ’不會增加製作程序。200538735 发明 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a cell counting classification wafer and a method for manufacturing the same. In particular, it refers to a cell counting method that uses an edge sheath flow to focus on the width of a sample fluid and uses etching techniques. Sort wafers. [Prior technology] The cell counting methods generally used in the biotechnology industry and academia are mostly using the cell counter proposed by Miyaky as shown in Fig. 1} Focusing a sample fluid with a side sheath flow, focusing the sample fluid to The width is only 1_ 10 15 20 The width through which the cells flow, and then count the number of cells in the sample fluid. In this detailed count B, one tube must be connected to the cell counter to form a sheath flow. In addition, four tubes are needed to add the sample fluid injection line U1 and the output fluid line 112. . Therefore, if multiple sample fluids need to be detected at the same time, more pipelines must be connected to form more side flow, which is inconvenient in testing and assembly. Referring to FIG. 2, since the ㈣counter 1 is a glass with multiple slots of different shapes of empty baskets, 12 ~ 16 are laminated to each other to form a plurality of hollow fluid channels, and the sample fluid and water are injected into the material. The water is formed into a side stream to squeeze and focus the sample fluid, and the fluid is leaked into the gap between the glass i2 to i6, and the production is also complicated. In addition, there are roughly two methods for the number of leaf cells. The first method is to use the Rong cursor to detect the cells that are stained as labels. The laser beam is used to emit the light, that is, the radiation is emitted after the laser light is emitted. Or the size, shape, and chemical composition of diffuse cells 6 200538735: In practical applications, the cell must accurately pass through the laser beam in the measurement area in the center a to ensure the maximum output signal. However, the above cell counting method requires the cells to be stained first, which is time-consuming and cumbersome to make, and the equipment used requires extremely high precision hydrodynamic and laser beams. Therefore, the cell counters and instruments used in this combination are very bulky. Large and expensive, the cost of use is high. ίο 15 ^ Another type of cell counter is to embed a pair of optical fibers axially corresponding to both sides of the tube and pass laser light at the end of a tube focused by the cell fluid. When cells pass through the optical fiber, the light between the cell and the two optical fibers, A change in the light intensity is said to be detected in the cell. And its fluid drive is to use integrated electrodes to drive the focus of cell flow ’so that the cells maintain the same height, and the intensity of the light signal caused by the cells changes. However, this technique requires elaborate procedures such as electrode and fiber alignment, and the manufacturing process is complicated. [Summary of the Invention] Therefore, the present invention provides a cell counting sorting wafer with simple fabrication, low leakage, and low fabrication cost, which can simultaneously classify multiple cell streams and classify them, and a method for fabricating the same. Another object of the present invention is to provide a cell detection method which is inexpensive to use and does not require expensive equipment. The cell counting classification wafer of the present invention is suitable for counting the number of cells in a sample fluid. The cell counting classification wafer includes a transparent base and a micro-channel unit hollowly formed in the base. The microchannel unit has a main channel, two subchannels arranged parallel to each other on two sides of the main channel, and a confluence channel connecting the main channel and the ends of the subchannels. The head end of the main channel communicates with an introduction hole formed on the surface of the base for the 20 200538735 sample fluid to enter. The head ends of the sub-channels communicate with each other and to an introduction hole formed by the water supply fluid formed on the surface of the base. The confluence channel is used to converge the sample fluid and the water fluid and communicate with an outflow hole formed on the surface of the base. When the water fluid in the two auxiliary channels flows to the confluence channel, a bilateral sheath flow is formed. The bilateral flow restricts the width of the sample fluid flowing out of the main pipe to the width that a single cell can circulate, and the cells flowing through are monitored by Quantity Calculates the number of cells contained in the sample fluid. The manufacturing method of the cell counting classification wafer includes the following steps: 15 (A) A main channel is etched on the top surface of a horizontal substrate, a secondary channel spaced horizontally on both sides of the main channel, and the head end is common. A bus channel connecting the main channel and the ends of the secondary channels, and the ends of the secondary channels communicate with each other. (B) Drilling holes are drilled through the heads of the main channels of the substrate to the bottom surface of the substrate through a drilling machine, and a common hole is drilled to the bottom surface of the substrate at the common connection of the head ends of the sub-channels. An injection hole, and an outflow hole drilled to the bottom surface of the substrate at the end of the busway. (C) A glass upper plate is covered on the top surface of the substrate and high temperature bonding is performed. The method for detecting cells using the "Hai cell juice number classification wafer" is to use a digital camera #camera to take an image to detect the cells in the sample counting fluid in the cell counting classification wafer, and the width of the sample fluid only allows one cell to flow alone. The steps of the method are as follows: (A) Using the digital camera: take a digital image of the sample fluid. (B) Circle one of the sample fluid flows in the digital image as the "first" area. (C) Chen Qi Measure the grayscale value of the shirt in the first area with the pretty Qi μ / dish. (D) Count the number of grayscale changes in the first region. The number of grayscale changes in the first region of the image in the first region 20 200538735 represents the number of cells flowing through. The effect of the present invention is to provide a simple structure of the device, save packaging and testing time, simplify the manufacturing process, and reduce the chance of gap leakage. 1 The number and type of cells can be detected, and cell counting and sorting wafers for cell sorting and recovery can be performed by applying dielectrophoretic force to the end of the 5 channels of the tube, and its manufacturing and use methods. ^ [Embodiment] The foregoing and other technical contents, features, and effects of the present invention will be clearly understood in the following detailed description of a preferred embodiment with reference to the accompanying drawings. Θ Referring to FIG. 3, FIG. 4, and FIG. 5, the cell counting classification wafer is used for counting the number of cells in the sample fluid 2. The cell counting classification wafer includes a transparent base 3, and a hollow space is formed on the base 3. The micro-channel unit 4 inside and an electrode unit 7 inserted into the micro-channel unit 4. 15 'Hai base 3 is made of transparent soda glass and is roughly flat. The Mayan test official road unit 4 has two horizontal hollow main passages 41, four auxiliary passages 42 arranged parallel to each other next to the main passage 41, and one connected to the ends of the 20A Tian J passages 41 and 42 and having a pipe diameter The narrowing width of the neck channel 43,-a water supply channel M connected to the head ends of the auxiliary channels 42, a confluence channel 45 connected to the end of the 込 43, and between the ends of the head of the confluence channel 45 and A first sorting channel, Bu 6 and a first sorting channel 47 respectively extending to the outside of the base 3 to the two sides. The four auxiliary channels 42 are two by two, and the pair of channels 42 are respectively arranged in parallel on two sides of a main channel 41. 9 200538735 The main channels 41 are connected to the main nozzle 411 of the neck channel 43, which is shrinking. The channel width is gradually becoming smaller. Cheng Yi, the field of livestock, and I 42 are connected to the neck channel 43: Decreasing width of the secondary pout 421. The neck channel is 43 j degrees; I, 41, 42 have a wider width. The visibility at 45 where the bus channel is connected is relatively rough, and a funnel shape is roughly formed. In this embodiment, the channel W and the four secondary channels 42 are used to analyze the two sample fluids 2 = 'in the top-the main channel 41 can be analyzed in conjunction with the second secondary channel C-one type, when multiple sample fluids 2 need to be analyzed at the same time, It can be applied by increasing the number of main channels and 15 20 sub-channels 42. Therefore, the implementation is not limited to the above-mentioned numbers... The inner peripheral surfaces of the head ends of the main channels 41 are respectively formed to penetrate the base. A guide hole 412 on the bottom surface of 3 is provided for the two sample fluids 2 to flow into the two main channels 41. An injection is formed on the inner peripheral surface of the head end of the water delivery channel 44 and penetrates to the bottom surface of the base 3. Hole 441, and branch into a plurality of branch sections 442, which branch sections 442 are connected to the head ends of the secondary channels 42, that is, the head ends of the four secondary channels 42 are commonly connected through the water conveyance channel 44, The water conveyance channel 44 is also used to guide the water fluid into the four auxiliary channels 42_. In this embodiment, the water fluid is deionized water, but as long as there is no obvious impurities in the water, ordinary water can be used as the water fluid. An inner peripheral surface of the end of the busway 45 is formed to penetrate to the bottom of the base 3 The sample fluid 2 and the water fluid are discharged out of the outflow holes 451 outside the base 3. The first and second classification channels 46 and 47 are respectively branched off from the branch channel 45 and extend to each of the three channels. A first classification hole 461 and a second classification hole 471 on the bottom surface of the base 3. 10 ί 15 20 200538735-The electrode unit 7 has-inserted into the refractory channel 45 adjacent to the first and second classification channels 46 and 47. The first electrode η at the communication point, an extension extending into the: a second electrode 72 in the classification channel 46 and extending to the first electrode, and a third electrode inserted in the busway 45 and extending to the first electrode; , And: a fourth electricity inserted in the second classification channel 47 and extending to the first electrode = 4. Thus, the electric secret is applied between the first and second electrodes 7 and 72 or the first 1 I71'73. Or between the first and fourth electrodes, the flow of silicon at the end of the M tube unit 4 / the melon body generates a dielectrophoretic force to drive different fines. 2 Flowing people "The end of the flow channel 45 or the first and second categories In channel 461 7. In the first embodiment, two classification channels are used as an example. The implementation can be achieved by using-^ knife channels. Referring to Figure 4 and Figure 6, Tian August uses the concept of flow diversion 1 a pump to push water fluid into the injection hole 44 丨 through the auxiliary channel 42 to spray four edges: two "to The four-way release side edge machine 422 enters the machine headway through the neck channel 43. Using this shunting principle 'and so on, you can push with: =, 51 on the same plane to get the required number of edge streams 422. No: the use of 51 miles also simplifies the packaging and testing time, and the flow velocity of the water fluid for 5 movements is stable. In the present embodiment, the implementation of the driving force of the fluid in the wafer using the screw pump is not limited to this. By this, the-the main channel 41, φ > Ding / main two kinds of different sample fluids 2, and the special secondary channel 42 is the pump and the main and secondary channels 41, 42 Although all the fluid of the peach body passes through the neck channel 43 and converges into the mouths 411 and 421, the water flowing out of the auxiliary channels 42 on each side of the main body when the convergence machine enters the confluence channel 45 is collected. The fluid forms a dihedral stream 422, 11 200538735 The dihedral stream 422 squeezes the σ and τ days like the fluid 2 squeezed in its center to form a fluid focusing effect. The flow rate ratio between the edge sheath flow 422 and the sample fluid 2 before passing through the field can reduce the flow rate of the sample fluid 2 to a few micrometers, which means that the two edge sheath flow 422 can Limit the width of the sample fluid 2 to a width that allows only a single cell to pass, and use a microscope (not shown) to count the two: the number of cells in the sample fluid 2. The above-mentioned water fluid is deionized water, but as long as there are no obvious impurities in the water, ordinary water can be used as the water fluid. However, with different types of liquid composition of water and fluid, the required speed ratio of the fluid ψ 隹 4 ′ claw-like coke will also be different. ίο 15 20 In addition, after counting in the sample stream 挤压 2 extruded by the side stream 422, a voltage can be applied between different electrodes of the electrode unit 7 to drive different cells in the sample fluid from different The channel is output outside the base 3. For example, when counting and observing the cells that need to be sorted and collected in the first sorting hole 461, an AC voltage may be applied between the first electrode 7m and the bakelite 72 to generate a positive dielectrophoretic force to attract the cells through the A sorting channel 46 is output to the first sorting hole 461 and collected outside the base 3. Using the same principle, different types of cells can be output to the base 3 through the classification holes 461, 471 or outflow holes 451, respectively, to facilitate recycling and reuse. In the description of the above example, the cell can also be classified by applying a negative dielectrophoresis force generated by applying electricity to repel the cells output from other channels, and the implementation is not limited to the above method. In addition, the main purpose of these sorting channels and electrode units 7 is to classify cells. In practice, the sorting channels and electrode units may not be provided, and still have the function of cell counting, so the scope of implementation is not limited to this. 12 200538735 For the detailed dimensions of each channel in the wafer of this embodiment, please refer to FIG. 4, wherein the width of the main nozzle 411 is 100 μm, the width of the sub channel 42 is 150 μm, and the width of the bus channel 45 is .24. Micron, and circle 6 is a photomicrograph of the sample fluid 2 in the confluence channel 45 that can be focused to a width of 5 degrees to 17 microns after adjusting the flow rate ratio. Referring to FIG. 7, the following continues to describe the manufacturing method of the cell counting classification wafer of the present invention: (A) A photoresist layer 33 is coated on the top surface of a horizontal glass substrate 31, and then the micrograph shown in FIG. 4 is drawn The photomask of the pattern of the channel system 4 covers the photoresist layer 33. The channel distribution is transferred to the substrate 31 by a developing technique, and the microchannel unit and the microchannel are etched with a glass etchant. The unit has been described in detail before and will not be repeated here. (B) Drill through the head end of the main channel 41 of the substrate 31 with a drilling machine-an introduction hole 412 penetrating to the bottom surface of the substrate 31, and drill a through hole at the head end of the water conveying channel 15 44 to An injection hole 々μ on the bottom surface of the substrate 31, an outflow hole 45 that penetrates to the bottom surface of the substrate 31 is drilled at the end of the busway 45, and φ is drilled at the ends of the first and second classification channels 46, 0 respectively. A first classification hole 461 and a second classification hole '471 are penetrated to the bottom surface of the substrate 31. Then use a mixed solution of sulfuric acid and thorium peroxide to boil for ten minutes. The substrate was cleaned and then dried. (C) Laying a layer of chromium (CO metal and a layer of gold (Au) metal) on the bottom surface of the glass upper plate 32 by evaporation, and then etch away the metal other than the electrode unit 7 by developing etching, and Use acetone, isopropyl alcohol (Is0-pr0pyl Alcohol, IPA), and deionized water to wash and dry sequentially. 13 200538735 Because the adhesion between the glass and the glass is not good, the metal interface is used to make the gold metal layer adhere well. (D) Position the 4 upper plate 32 on the top surface of the substrate, and in a high temperature sintering furnace filled with nitrogen, heat the upper plate M and the substrate 31 at a high temperature to make the glass appear in a heterogeneous state and join. So that the channels are formed hollow between the substrate 31 and the upper plate 32. High-temperature bonding in a nitrogen environment can prevent the metal electrodes from oxidizing and causing the wafer to be damaged. (E) The upper plate 32 and the substrate 31 will be bonded. After encapsulation, a cell counting classification wafer is formed. Ο The cell counting classification wafer produced by this manufacturing method has the following advantages: 32 15 〇) Only a glass substrate 31 and an upper plate, phase Splicing joining temperature alone, (iv) five glass structure to hide engagement ratio of conventional nip between the glass plates to reduce the chance of leakage _, simplified production process. ⑺This production method can produce multiple films that can simultaneously detect multiple sample fluids 2 ′ It is only necessary to use a mask 34 with different channel structures in the shadow process, and it will not increase the production process.
20 本發明之另-目的在於提供使用上述細胞計數分類晶 片的方法’參閱圖8，其係於該細胞計數分類晶片内之兩種 樣品流體2在流體聚焦後，配合—數位攝影機52快速擷取 ㈣’ J1L里單元53上處理拍攝之影像，並由影像處 理中檢測細胞。該細胞檢測方法之步驟如下： (Α)以所述數位攝影機52拍攝所述樣品流體2之寬 度為僅容單-細胞通過的寬度時流動之數位影像，數位攝 14 200538735 影機52須具有—朝向該晶片之匯流通道45中樣品流體2 的顯微鏡頭，以使攝影的倍率放大，將細胞放大到肉眼可 見的大小，且數位攝影機52輸出一影像訊號。 (B) 以一處理單元53接收該數位攝影機所輸出之 〜像並解析出複數影像圖片。本實施例之數位攝影 機52是採用每秒94張圖片來拍攝’也就是由該影像訊號 每心可解析出94張景彡像圖片。且所擷取影像為灰階圖片， 以利細胞影像之判別。該處理單元53是—般常見之電腦， 但實施上不以上述之方式為限。 ίο 15 20 (C) 於5亥等影像圖片中該二樣品流體2之流程中各選 擇一區域61。 (ιυ監測該二區域61巾之影像灰階值。數位攝影機 拍攝之影像為灰階值，在影像中細胞粒子在灰階值中呈 現偏黑狀態，與背哥的值ώ “、的偏白上有相當程度的對比。利用此 原理’ S細胞通過該區域Μ時4接 邊線，邊線上合因為"… 域61之兩侧 的灰階值比較黑，與背景灰階值 通過。 ，以此影像變化分析，而可認定-細胞粒子彳 (/)計數該二區域61之影像灰階值改變次數。該二 數之兩側邊線影像灰階值改變之次數就代表細胞流過 之數目。以藉此計數該二樣品流體2内之細胞數量。 參閲圖9，利用上述之方式也 胞粒子來進甘 切用來針對不同大小的細 =订计數。其只須在影像上之每一樣品流體2於 4道45的流程中同時圈選出不同大小之長方形的-第 15 200538735 區域62及一第 區域63並分別對第 5 10 15 20 ^ ……1不-、二區域62、63 7數即可。舉例說明如下：如果注人包含兩種不同大小細 ι粒子的樣品流體2進人主通道41中，而兩種細胞大小分 =為20微米與12微米，則把該第-、二區域62、63寬度 ^別定義為20微米肖12微米大小，並定義該等區域的: 線必須同時偵測到灰階變化才能計數得一細胞粒子。者 '2微米的細胞通過寬度2〇微米的第一區域時不可： 域62的兩邊’所以第-區域62未伯測12 田I。當12微米的細胞通過寬12微米的第二區域 63時，其兩邊線可同時伯測到變化，所以第二區域幻伯測 ，計數一細胞通過。當20微米的細胞出現時，第一及第二 區域62、63的兩邊線都可㈣仙到影像灰階值變化 以兩個區域皆會谓測並計數一細胞通過。計數過程之後， ^圈^的第一區域62計數所得的細胞數目即為真正20微 二^的數目’而第二區域63計數所數得之細胞數目同時 =微米及2。微米的細胞數，所以真正Η微米的細胞 二區域63計數測得數字減掉2。微米細胞數即為《 真的12微米的細胞數目。由此可知，應用上述之方 測得兩種不同大小之 Μ 、，，^數目，也就是說此方法可偵測到 數種不同種類之細胞數目。 另^卜應用數位影像谓測方法亦可測得該樣品流體2之 二的’⑼速方法是先在影像上之樣品流體2難流通道 二王中圈選二區$ 64，並㈣測細胞之大小定義該二 區域64之尺寸士丨 L 。此時由影像中可得知兩區域64間隔 16 ίο 15 20 200538735 實際距離，並可由影片之數旦 〈数里侍知一細胞通過該二區域64 之時間差，以量測得之味M ¥ ^ 、、 夺間差和距離，即可算得該樣品流 體2之流速。该二區域64問夕阳施叮/ a 4間之距離可依貫際樣品流體2中 之細胞密度量訂定，以準磁旦 確里取一、、、田胞流經該二區域64間 之時間。 運用上述之檢測方法，不僅可同時計數兩種樣品流體2 之細胞數量，並可量測得不同大小之細胞數量，及流體之 速度。 m述，本發明利用利用分流之觀念，僅以一果浦^ 就可驅動水流體於四副通道中流動形成四條均句邊勒流， =備構造簡單，並節省封裝、測試時間，更可使推動之水 流體流速平穩。其次是利用姓刻技術製作該晶片，簡化製 作知序i避免縫隙攻漏之機會。最後利用侦測影像灰階 ，變化的方法’可同時_樣品流體中之不同種類之細胞 數目及樣品流體之流速。以構造簡易之裝置達咖細胞 之功用，故確實能達到發明之目的。 惟以上所述者，僅為本發明之較佳實施例而已，當不馨 ，以此限定本發明實施之範圍，即大凡依本發明申請專利 範圍及發明說明書内容所作之簡單的等效變化與修姊，皆 應仍屬本發明專利涵蓋之範圍内。 【囷式簡單說明】 圖1是習知之細胞計數器之立體圖，其中最上層之玻 璃板已掀除； 圖2該習知之細胞計數器之立體分解圖； 17 200538735 圖3本發明細胞計數分類晶片 _ m ^佳貫施例的立體分 解圖； 圖4是該較佳實施例之一微管道單元的俯視圖，說明 各通道之實際規袼； 圖5是該較佳實施例之—匯流通道與二分類通道連接 處之局部放大圖，說明一電極單元之設置位置； 圖6是該較佳實施例之匯流通道中一樣品流體可聚焦 至寬度達17微米之顯微照片； 圖7是本發明細胞計數器之製造過程示意圖； 圖8疋本發明細胞計數器以一泵浦推動邊鞘流並與一 數位攝影機及一處理單元之架構示意圖，說明由一樣品流 體中擷取影像計數細胞的方法；及 圖9是該較佳實施例之複數邊鞘流聚焦該樣品流之示 忍圖’ 5兒明影像擷取分析不同細胞數量及樣品流體之速度 的方法。 18 200538735 【圖式之主要元件代表符號說明】 2 樣品流體 451 流出子L 3 基座 46 第一分類通道 31 基板 461 第一分類孔 32 上板 47 第二分類通道 33 光阻劑層 471 第二分類孔 34 光罩 51 泵浦 4 微管道單元 52 數位攝影機 41 主通道 53 處理單元 411 主喷嘴 61 區域 412 導入孔 62 第一區域 42 副通道 63 第二區域 421 副喷嘴 64 區域 422 邊鞠流 7 電極單元 43 頸通道 71 第一電極 44 輸水通道 72 第二電極 441 注入孔 73 第三電極 442 分歧段 74 第四電極 45 匯流通道 1920 Another aspect of the present invention is to provide a method for using the above-mentioned cell counting classification wafer. Referring to FIG. 8, the two sample fluids 2 in the cell counting classification wafer are in focus after the fluid is focused, and the digital camera 52 quickly captures ㈣ 'J1L processes the captured image on unit 53 and detects cells during image processing. The steps of the cell detection method are as follows: (A) The digital camera 52 is used to capture a digital image of the sample fluid 2 flowing when the width of the sample fluid 2 is only allowed to pass by the cell. The digital camera 14 200538735 Toward the microscope head of the sample fluid 2 in the confluence channel 45 of the wafer, the imaging magnification is enlarged, the cells are enlarged to the size visible to the naked eye, and the digital camera 52 outputs an image signal. (B) A processing unit 53 receives the ~ image output by the digital camera and parses out multiple image pictures. The digital camera 52 of this embodiment uses 94 pictures per second for shooting ', that is, 94 images of sceneries can be parsed from the image signal per heart. In addition, the captured image is a gray-scale image to facilitate the identification of cell images. The processing unit 53 is a common computer, but the implementation is not limited to the above manner. ίο 15 20 (C) Select a region 61 in the process of the two sample fluids 2 in the images such as May. (ιυ monitors the grayscale value of the 61 towels in the two areas. The image captured by the digital camera is a grayscale value. In the image, the cell particles appear black in the grayscale value, which is inferior to the value of the older brother. There is a considerable degree of contrast. Using this principle, the S cell passes 4 edges when passing through the area M. The edges are black because the grayscale values on both sides of the domain 61 pass through, and pass through the background grayscale values. Based on this image change analysis, it can be determined that the cell particle 彳 (/) counts the number of grayscale changes in the image of the two regions 61. The number of grayscale changes in the edge image on both sides of the two represents the number of cells flowing through. In order to count the number of cells in the two sample fluids 2. Referring to FIG. 9, the above-mentioned method is also used to enter the cells to cut and count for different sizes of fine particles. It only needs to be on the image. For each sample fluid 2 in the flow of 4 and 45, circled rectangles of different sizes at the same time-15th 200538735 area 62 and a first area 63 and respectively for the 5th 10 15 20 ^ ...... 1 no-, two areas 62, 63 7 can be counted. An example is as follows: The sample fluid 2 containing two different-sized fine particles 2 enters the main channel 41, and the two cell sizes are 20 microns and 12 microns. The widths of the first and second regions 62 and 63 are defined as 20, respectively. Micron Shaw 12 micron size, and define these areas: the line must detect grayscale changes at the same time in order to count a cell particle. '2 micron cells cannot pass through the first region of 20 micron width: Domain 62 of On both sides, so the first-region 62 has not been measured 12 fields I. When a 12-micron cell passes through a second region 63 that is 12 micrometers wide, the two sides can simultaneously detect changes, so the second region is magically measured and counted. One cell passes. When 20 micron cells appear, both sides of the first and second regions 62 and 63 can be changed to the grayscale value of the image. In both regions, a cell passes and is counted. Count After the process, the number of cells counted in the first area 62 of the circle ^ is the true number of 20 microseconds' and the number of cells counted in the second area 63 is equal to both microns and 2. The number of cells in microns, so The number of 63 counts in the real two micron cell area was reduced 2. The number of micron cells is the number of cells that are really 12 microns. It can be seen that the number of two different sizes of M,, and ^ is measured by the above method, which means that this method can detect several different The number of types of cells. In addition, the digital fluid pre-measurement method can also be used to measure the sample fluid 2 bis' speed method is first on the image of the sample fluid 2 difficult flow channel two kings in the second circle $ 64 , And the size of the cell is defined to define the size of the two regions 64. L. At this time, the actual distance between the two regions 64 can be known from the image. 15 20 200538735 The flow rate of the sample fluid 2 can be calculated by measuring the time difference between the time when the cells pass through the two regions 64 and the measured flavors M ¥ ^,, and the distance between them. The distance between the 64 areas of the two areas and the setting sun / a 4 can be determined according to the cell density in the sample fluid 2. The field cells are taken through the quasi-magnetic field to flow through the 64 areas of the two areas. time. By using the above detection method, not only the number of cells in two sample fluids 2 can be counted at the same time, but also the number of cells of different sizes and the velocity of the fluid can be measured. As described above, the present invention utilizes the concept of using a shunt, and can drive water to flow in four pairs of channels to form four uniform sentence edges with only one Guopu ^, which is simple in construction and saves packaging and testing time. Make the flow velocity of the pushing water fluid stable. The second is to use the last name engraving technology to make the chip, simplify the production process, and avoid the opportunity of gaps. Finally, the method of detecting image gray levels is used to change the number of different types of cells in the sample fluid and the flow rate of the sample fluid. The function of the cells is achieved by a simple device, so it can indeed achieve the purpose of the invention. However, the above are only the preferred embodiments of the present invention. When unsatisfactory, the scope of the implementation of the present invention is limited, that is, the simple equivalent changes made according to the scope of the patent application and the content of the invention specification of the present invention. Sisters, should still fall within the scope of the invention patent. [Brief description of the formula] Figure 1 is a perspective view of a conventional cell counter, in which the top glass plate has been removed; Figure 2 is an exploded view of the conventional cell counter; 17 200538735 Figure 3 Cell counting classification chip of the present invention_ m ^ Three-dimensional exploded view of the preferred embodiment; Figure 4 is a top view of a micro-channel unit of the preferred embodiment, illustrating the actual rules of each channel; Figure 5 is the preferred embodiment-the convergence channel and two classification channels A partially enlarged view of the connection, illustrating the placement of an electrode unit; Figure 6 is a photomicrograph of a sample fluid in the confluence channel of the preferred embodiment that can be focused to a width of 17 microns; Figure 7 is a micrograph of the cell counter of the present invention Schematic diagram of the manufacturing process; Figure 8: Schematic diagram of the cell counter of the present invention using a pump to drive the side sheath flow and a digital camera and a processing unit, illustrating a method for capturing images to count cells from a sample fluid; and Figure 9 is The method of capturing the sample stream by analyzing the number of cells and the velocity of the sample fluid in the preferred embodiment of the multiple edge sheath flow focusing diagram of the sample flow. 18 200538735 [Description of the main symbols of the diagram] 2 Sample fluid 451 flows out of sub-L 3 base 46 first classification channel 31 substrate 461 first classification hole 32 upper plate 47 second classification channel 33 photoresist layer 471 second Sorting hole 34 Photomask 51 Pump 4 Micro-channel unit 52 Digital camera 41 Main channel 53 Processing unit 411 Main nozzle 61 Area 412 Introduction hole 62 First area 42 Sub channel 63 Second area 421 Sub nozzle 64 Area 422 Side flow 7 Electrode unit 43 neck channel 71 first electrode 44 water delivery channel 72 second electrode 441 injection hole 73 third electrode 442 branching section 74 fourth electrode 45 busway 19