1269035 玖、發明說明： 【發明所屬之技術領域】 綱是有關於一種細胞計數分類晶片及其製造方法 ’特別是指-種利用邊鞘流聚焦樣品流體寬度且❹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！所示的由Miyaky所提出之細胞計數器ι以邊鞠流 聚焦一樣品流體的方式，將該樣品流體聚焦至寬度僅容一籲 1〇 細胞流過的寬度，再計數該樣品流體中之細胞數量。該細 胞計數晶片中形成-邊勒流就須接引一管路ιι〇至該細胞計 數益’再加上注入樣品流體之管路m &輸出流體之管路 112就需四條管路。因此，如須同㈣測多種樣品流體時， 須接引更多之管線以形成更多之邊鞘流，於測試及組裝上 15 都不方便。 參閱圖2，由於該細胞計數器1是以複數刻有不同形狀 之簍空槽孔的玻璃片12〜16互相疊貼以形成複數中空之流_ 體通道，並注入樣品流體及水進入該等通道，使水形成邊 鞘ML夾細聚焦該樣品流體，容易造成玻璃板12〜ι6間缝隙， 〇 漏洩流體，且於製作上也較為繁雜。 另外’細胞計數之方法大略有兩種，第一種是利用勞 光‘疋來檢測經染色做為標籤的細胞，以雷射集中光束於 聚焦後之樣品流體上，藉由細胞照射雷射光後放射或是散 射出來的光’即可知道細胞的大小、形狀及化學組成成份 1269035 、:在實際的應用時’細胞必須準確地經過量測區域的雷射 先束正中央，以確保能得到最大的輸出訊號。但上述之细 =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 方式所組合的細胞計數器及儀器設備體積都很龐 昂貴，使用成本較高。 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侧的光纖並通入雷射光，利 用細胞經過光纖時，細胞阻斷二光纖間之光線，引起光線籲 的變化來偵測到細胞。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 號變化強度一致。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’本發明即在提供一種製作簡易、不易茂漏及低 製作成本且可同時偵測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鲁 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、一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 7 1269035 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，該二邊鞘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： (A)於一水平之基板之頂面上蝕刻出一主通道、一平鲁 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 一貫穿至該基板底面之導入孔，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 道之末立而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。 而使用該細胞計數分類晶片檢測細胞之方法係使用一 _ 數位攝影機擷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 過’該細胞檢測方法之步驟如下：（A)以所述數位攝影機 拍攝所述樣品流體流動之數位影像。（B)目選該數位影像 中樣品流體之流程中的一小段落作為一第一區域。（c^監 測該第—區域中之影像灰階值。（D)計數該第_區域之影 像灰階值改變次數，該第一區域中影像灰階值改變之次數 1269035 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，且減低縫隙茂漏之機會。' 益可偵測細胞數目及種類，且可藉著施加介電泳力於微管 5 道單疋末端進行細胞分類回收的細胞計數分類晶片及其製 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，將可清 IQ 楚的明白。 參閱圖3、圖4與圖5，該細胞計數分類晶片是用於計 數複數樣品流體2内之細胞數量，該細胞計數分類晶片包 括一透明之基座3、一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 該基座3是由透明之鈉玻璃材質製作而成，概略成水 平之板狀。 该微管道單元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 等主、副通道41、42末端並管徑寬度漸縮的頸通道43、一 2〇 接績連通該等副通道42之頭端的輸水通道44、一連通於該 之兩側。 頸通道43末端的匯流通道45，及連通於該匯流通道45頭 、末端間並分別向兩側延伸至該基座3外的一第一分類通 道46與一第二分類通道47。該四副通道42是兩兩成對， 每一對副通道42是分別平行排列於一主通道41 9 1269035 該等主通道41於連通該頸通道43處各形成一通道寬度漸 縮的主喷嘴411，該等副通道42於連通該頸通道43處亦各 形成一通道寬度漸縮的副噴嘴421。該頸通道43速通該等 主、副通道41、42處寬度較寬，接通該匯流通道45處之 5 寬度較窄，概略形成一漏斗形狀。於本實施例中是以二主 通道41及四副通道42以分析兩種樣品流體2内之細胞做 說明，實際上一主通道41配合二副通道42就可分析一種 樣品，需同時分析多種樣品流體2時，就增加主通道41及 副通道42之數量即可應用，因此實施上不以上述之數量為_ 10 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之底面的導入孔412，以分別供所述兩種樣品流體2流 入该二主通道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# 本貝％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疋分別由該匯流通道45岔流分支出來，並分別延伸 至形成於該基座3底面的一第一分類孔461與一第二分類 孔 471 〇 10 1269035 5 10 15 該電極單元7具有—插伸於該匯流通道45内鄰近 -、二分類通道46、47連通處的第_電極71、—播伸二 第-分類通道46内並往第-電極延伸的第二電極 伸於該匯流通道45内並往第-電極延伸的第三電極^ Z -插伸於該第二分類通道47内並彳n極延伸的第四電 極74。藉此，施加電壓於第一與第二電極71、72間或第— 與第三電極71、73間，或第一與第四電極71、Μ間，Z 於該微管道單it 4末端之流體產生介電泳力驅動不;之= 胞分別流入該匯流通道45末端或該第一、第二分類通道2 、47内。本實施例中是以二分類通道作說明，實施上ζ 一 分類通道就可達成分類之功效。 參閱圖4及圖6,本發明利用分流之觀念，以一個栗浦 51推動水流體進入注入孔441，經由輸水通道44分流至四 副通道42,以噴流四條均勻邊鞘流似經頸通道43進入匯 流管道。使用此分流原理，以此類推，即可在同一平面以 -個泵浦51推動水流體以獲得所需的邊鞍流422數目。不 僅節省泵浦51數量之使用，也解省封裝及測試之時間，更_ 可使推動之水流體流速平穩。本實施例中是以蠕動式泵浦 51作為晶片内流體之驅動力，實施上不以此為限。 藉此，該二主通道41注入兩種不同之樣品流體2，而 該等副通道42是由該泵浦51注人水流體，#所有流體經 由該主、副通道41、42之主、副噴嘴411、421後，匯集喷 入該頸通道43，並匯流進入該匯流通道45中時，每一主通 道41之兩側副通道42中流出之水流體形成二邊鞘流422， 20 1269035 5 10 15 20 該二邊鞠流1 形成流體聚焦效應，此時適當地調整邊鞘流422與樣品流 體2之流速比可將樣品流體2之寬度縮減至數微米寬，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）就可計數所述樣 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，流體聚焦的所需的速度比亦會 不一樣0 ! 此外，經過邊鞘流422擠壓之樣品流體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 λ ,，外例如，經计數觀察後之細胞如須分類收集至第 一分類孔461時，可施加一交流電壓於該第一電極71與第 電才° 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-分類孔461輪出至該基座3外收集。運 用相同之原理就可驅動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’亦可用施加電壓產生負介電泳力以 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 分類細胞而設置’於實施上亦可不設置分類通道 ”包極單兀’仍然具有細胞計數之功能，因此實施範圍不 以此為限。 野1开爽於其中央之樣品流 12 1269035 於本實施例之晶片中各通道之詳細尺寸請參考圖4，其 中該主噴嘴411之寬度為1〇〇微米，該副通道42之寬度為 15〇微米，而匯流通道45之寬度為24〇微米，而圖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Μ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_ 1〇 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4。 該微管道單元已於前詳述，不再贅述。 (B) 在该基板31之該等主通道41之頭端端以鑽孔機 鑽出貝牙至該基板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441、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45i，及分別於該第一、第二分類通道46、47末端鑽出籲 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氫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) 於一玻璃上板32之底面上以蒸錄的方式先後舖 设一層鉻（Cr )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（Iso-Propyl AlcohoUPA )、去離子水循序沖洗並烘乾。 13 1269035 因為金與玻璃黏著性不好，所以利用鉻 W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。 5 10 15 20 (D) 把該上板32 ^位覆蓋於該基板3ι頂面並於一充 滿氮氣環境之高溫燒結爐中’彻高溫加熱上才反Μ及基板 31使玻璃呈現融熔狀態而接合，以使該等通道中空地形成 於該基板3i及上板32 ι在氮氣環境下進行高:接合可 避免金屬電極發生氧化而造成晶片損毁。 (E) 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。 以本製作方法製作出細胞計數分類晶片具有以下之優 (1) 僅以一蝕刻該等通道後之玻璃基板31及一上板 32相互璺接並以高溫接合，&習知五玻璃基板疊合之結構 ，減少玻璃板間之隙縫洩漏之機會，也簡化製作程序。 (2) 本製作方法可製作出可同時檢測多種樣品流體2 之a曰片/、須於顯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。 本發明之另一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，配合一數位攝影機52快速擷取 圖片，並於一處理單元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： (A)以所述數位攝影機52拍攝所述樣品流體2之寬 度為僅容單一細胞通過的寬度時流動之數位影像，數位攝 14 1269035 影機52須具有一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52所輸出之 影像訊號，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 每秒可解析出94張影像圖片。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。 | (C) 於該等影像圖片中該二樣品流體2之流程中各選 擇一區域61。 (D) 監測該二區域61中之影像灰階值。數位攝影機 52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’與背景的偏白上有相當程度的對比。利用此 原理’當細胞通過該區域61時會接觸到該區域6丨之兩側 邊線邊線上會因為細胞的灰階值比較黑，與背景灰階值 車乂白有所不同，以此影像變化分析，而可認定-細胞粒子_ 通過。 (E) 计數該二區域61之影像灰階值改變次數。該二 品或61之兩側邊線影像灰階值改變之次數就代表細胞流過 妻目以猎此计數該二樣品流體2内之細胞數量。 ”參閱圖9，利用上述之方式也可用來針對不同大小的細 已粒子來進行汁數。其只須在影像上之每一樣品流體2於 匯流通道45的流程中同時圈選出不同大小之長方形的一第 15 5 10 15 20 1269035 吁^ 區域63並分別對第―、二區域62、63 的卩可。舉例說明如下：如果注入包含兩種不同大小， 胞粒子的樣品流體2進人主通道41中 幻細 纪丨丨达， Τ 而兩種細胞大小分 別為20微米與12微米，則把該、- 分別中μ & — &域62、63寬度 邊续義為20微米與12微米大小，並μ該等區域的兩 纽必須同時損測到灰階變化才能計數得—細胞粒子。去 微未的細胞通過寬度20微米的第—區域62時 : ::ΓΓ區域62的兩邊，所以第一區域62未_:; …田肊。當12微米的細胞通過寬12微米的第二區域( 63 其兩邊線可同時偵測到變化，所以第二區域偵測 並汁數—細胞通過。當20微米的細胞出現時，第—及第二 區域62、63的兩邊線都可同時偵測到影像灰階值變化，戶： 以兩個區域皆會_並計數—細胞通過。計數過程之後， 戶斤圈選的第-區域62計數所得的細胞數目即為真正2〇微 米細胞的數目’而第二區域63計數所數得之細胞數目同時 包含12微米及20微米的細胞數，所以真正12微米的細胞 數目是第二區域63計數測得數字減掉2()微米細胞數即為| 真正的12微米的細胞數目。由此可知，應用上述之方法可 測得兩種不同大小之細胞數目’也就是說此方法可偵測到 數種不同種類之細胞數目。 另外應用數位影像偵測方法亦可測得該樣品流體2之 流速，其測速方法是先在影像上之樣品流體2於匯流通道 45的流程中圈選二區域64，並依偵測細胞之大小定義該二 區域64之尺寸大小。此時由影像中可得知兩區域64間隔 16 1269035 實際距離，並可由影片之數 之時間差，以量測得之時:::知一細胞通過該二區域64 體2之r + 、B差和距離，即可算得該樣品流 篮2之流速。該二.區域64間夕呢她π n c…一 離可依實際樣品流體2中 之細胞禮度：Ε盯定，以準墟旦 之時間。 旱確里取一細胞流經該二區域64間 運用上述之檢測方法，不僅 ^ , Η, ^ ^ 1胃 个惶j Π時叶數兩種樣品流體21269035 玖, invention description: [Technical field to which the invention pertains] The present invention relates to a cell counting and classifying wafer and a method of manufacturing the same, in particular, a cell counting using a side sheath flow to focus a sample fluid width and a cutting technique Sort the wafer. [Prior Art] Most of the cell counting methods commonly used in the biotechnology industry and in the academic world are as shown in the figure! The cell counter ι proposed by Miyaky is shown to focus the sample fluid in a turbulent manner, focusing the sample fluid to a width that allows only one cell to flow, and then counting the number of cells in the sample fluid. . In the cell counting wafer, the formation of a side stream requires the introduction of a pipe to the cell count, plus the pipe for injecting the sample fluid m & the outlet fluid line 112 requires four lines. Therefore, if it is necessary to test a plurality of sample fluids with (4), more pipelines must be connected to form more side sheath flows, which is inconvenient for testing and assembly. Referring to FIG. 2, since the cell counter 1 is stacked with a plurality of glass sheets 12 to 16 having different shapes of hollow slots, a plurality of hollow flow channels are formed, and sample fluid and water are injected into the channels. The water is formed into the side sheath ML to focus the sample fluid, which easily causes a gap between the glass plates 12 to ι6, which leaks fluid, and is complicated in production. In addition, there are two methods for 'cell counting. The first one is to use Luguang's to detect cells stained as labels, and concentrate the beam on the focused sample fluid by laser irradiation. Radiation or scattered light 'can know the size, shape and chemical composition of the cell 1269035,: In practical applications, the cell must accurately pass through the center of the laser beam in the measurement area to ensure maximum Output signal. However, the above-mentioned fine=counting method needs to dye the cells first, which is time consuming and troublesome, and the use of the device requires extremely high precision fluid dynamics and laser beams, so the cell counter and the instrument equipment combined by this method are very bulky. Pang is expensive and the cost of use is high. Another type of cell counter is buried in a pair of tubes at the end of the tube where the fluid is focused, and a pair of optical fibers corresponding to the two sides of the tube are inserted into the laser light. When the cells pass through the optical fiber, the cells block the light between the two fibers, causing light. Call for changes to detect cells. The fluid drive is to integrate the electrodes to drive the cell flow to focus, so that the cells maintain the same height, and the intensity of the light signals caused by the cells is consistent. However, this technology requires precise procedures such as electrode and fiber alignment, and the process is complicated. SUMMARY OF THE INVENTION Accordingly, the present invention provides a cell counting and classifying wafer which is easy to manufacture, easy to leak, and low in production cost, and which can simultaneously detect and classify a plurality of cell streams, and a method of fabricating the same. Another object of the present invention is to provide a cell detection method which is inexpensive to use and which does not require expensive equipment. The cell counting and sorting wafer of the present invention is adapted to count the number of cells in a sample fluid, the cell count sorting wafer comprising a transparent pedestal, and a micro-pipe unit hollowly formed in the susceptor. The micro-pipe unit has a main channel, a sub-channel arranged on two sides of the main channel in parallel with each other, and a confluence channel communicating with the end of the main channel and the sub-channels. The head end of the main passage is connected to an introduction hole formed in the surface of the base for the entry of the fluid of the 7 1269035 sample. The head ends of the sub-channels communicate with each other and are connected to an introduction hole formed in the surface of the base to which the water supply fluid enters. The manifold channel is for confluently drawing the sample fluid and the water fluid and communicating to an outflow aperture formed in the surface of the base. When the water in the two sub-channels flows to the confluence channel, a two-sided sheath flow is formed, the two-sided sheath flow restricting the width of the sample fluid flowing from the main pipe to a width circulated by a single cell, and monitoring the cells flowing through The quantity counts the number of cells contained in the sample fluid. The method for manufacturing the cell count classification wafer comprises the following steps: (A) etching a main channel on a top surface of a horizontal substrate, a sub-channel separated by a flat line on both sides of the main channel, and the head end co-connecting the main The channel and a confluence channel at the end of the sub-channels, the head ends of the sub-channels being in communication with each other. (B) drilling a through hole into the bottom surface of the substrate by a drill at the head end of the main channels of the substrate, and drilling a through hole to the bottom surface of the substrate at a common communication between the head ends of the sub-channels An injection hole is formed, and an outflow hole penetrating through the bottom surface of the substrate is drilled at the end of the bus passage. (C) A glass upper plate is placed on the top surface of the substrate and joined at a high temperature. The method for using the cell count sorting wafer to detect cells is to detect the cells of the sample fluid in the sorting wafer by using a digital camera to capture images, and the width of the sample fluid is only allowed to flow through a single cell. The steps of the cell detection method are as follows: (A) taking a digital image of the sample fluid flow with the digital camera. (B) A small section in the flow of the sample fluid in the digital image is selected as a first region. (c) monitoring the grayscale value of the image in the first region. (D) counting the number of times the grayscale value of the image in the first region is changed, and the number of times the image grayscale value is changed in the first region is 1269035, which represents the flow of cells. The utility model can provide an apparatus with simple structure, saves packaging, test time, simplifies the production process, and reduces the chance of gap leakage. ' The number and type of cells can be detected, and the dielectric can be applied A cell count sorting wafer for performing cell sorting and recovery at the end of a single tube of microtubules, and a method for manufacturing and using the same. [Embodiment] The foregoing and other technical contents, features, and effects of the present invention are described below with reference to the drawings. In the detailed description of a preferred embodiment, it will be clear from the IQ. Referring to Figures 3, 4 and 5, the cell count sorting wafer is used to count the number of cells in the plurality of sample fluids 2, the cell count classification The wafer includes a transparent base 3, a micro-pipe unit 4 hollowly formed in the base 3, and an electrode unit 7 inserted into the micro-pipe unit 4. 15 The micro-pipe unit 4 has two horizontal hollow main passages 41, four sub-channels 42 arranged parallel to each other at the side of the main passage 41, and one continuous connection. The neck passages 43, the ends of the main and auxiliary passages 41, 42 and the tube diameter are tapered, and the water passages 44 that communicate with the head ends of the auxiliary passages 42 are connected to the sides. a first flow channel 45 and a second sorting channel 47 extending from the head end of the channel 43 and extending between the ends of the channel 45 and extending to the outside of the base 3, respectively. 42 is paired in pairs, each pair of sub-channels 42 are respectively arranged in parallel in a main channel 41 9 1269035. The main channels 41 form a channel-width tapered main nozzle 411 at the direction connecting the neck channels 43. The sub-channels 42 also form a sub-nozzle 421 having a tapered width at the passage of the neck passages 43. The neck passages 43 are open to the main and sub-channels 41, 42 at a wide width, and the converging passages 45 are opened. The width of the 5 is narrow and roughly forms a funnel shape. In the embodiment, the two main channels 41 and the four sub-channels 42 are used to analyze the cells in the two sample fluids 2. In fact, one main channel 41 can be combined with the two sub-channels 42 to analyze one sample, and multiple sample fluids need to be analyzed simultaneously. At 2 o'clock, the number of the main channel 41 and the sub-channel 42 can be increased, so that the number of the main channel 41 and the sub-channel 42 can be applied. Therefore, the number of the main channels 41 is not limited to _10. The inner peripheral surfaces of the main channels 41 are respectively formed to penetrate the base. The introduction hole 412 of the bottom surface of the seat 3 is configured to respectively supply the two sample fluids 2 into the two main channels 41. An inner injection surface of the head end of the water delivery channel 44 forms an injection through the bottom surface of the base 3. The hole 441 is branched and branches out of the plurality of divergent segments 442. The divergent segments 442 are connected to the head ends of the sub-channels 42, that is, the head ends of the four sub-channels 42 are connected by the water passage 44. The water passage is also guided by the water passage 44 to guide the water into the four passages. The water fluid is deionized water, but ordinary water can be used as the water fluid as long as there is no obvious impurity in the water. An inner peripheral surface of the end of the bus passage 45 is formed with an outflow hole 451 penetrating the bottom surface of the base 3 to discharge the sample fluid 2 and the water flow out of the base 3. The first and second sorting channels 46 47 are respectively branched and branched by the collecting channel 45, and respectively extend to a first sorting hole 461 and a second sorting hole 471 〇 10 1269035 formed on the bottom surface of the base 3 respectively. 5 10 15 The electrode unit 7 has a first electrode 71 embedded in the confluence channel 45 adjacent to the -, two-class channels 46, 47, and a second extension-classification channel 46 extending toward the first electrode. The third electrode extending in the bus passage 45 and extending toward the first electrode is inserted into the fourth electrode 74 of the second sorting passage 47 and extending in the n pole. Thereby, a voltage is applied between the first and second electrodes 71, 72 or between the first and third electrodes 71, 73, or between the first and fourth electrodes 71, Μ, Z at the end of the micro-pipe single it 4 The fluid generates dielectrophoretic force to drive no; the cells = flow into the end of the confluence channel 45 or the first and second sorting channels 2, 47, respectively. In this embodiment, the two classification channels are used for description, and the classification effect can be achieved by implementing the upper classification channel. Referring to FIG. 4 and FIG. 6, the present invention utilizes the concept of shunting to push a water fluid into the injection hole 441 by a Lipu 51, and divert it to the four sub-channels 42 through the water delivery passage 44, so that four uniform side sheath flows like a neck passage through the jet flow. 43 Enter the confluence pipe. Using this shunting principle, and so on, the water fluid can be pushed by a pump 51 in the same plane to obtain the desired number of side saddles 422. Not only does it save the use of pump 51, it also saves time for packaging and testing, and it also smoothes the flow rate of the propelled water fluid. In the present embodiment, the peristaltic pump 51 is used as the driving force of the fluid in the wafer, and the implementation is not limited thereto. Thereby, the two main channels 41 inject two different sample fluids 2, and the sub-channels 42 are filled with the water fluid by the pump 51, and all the fluids pass through the main and auxiliary channels 41, 42 After the nozzles 411 and 421 are collected and injected into the neck passage 43 and merged into the manifold passage 45, the water flowing out from the secondary passages 42 on both sides of each main passage 41 forms a sheath flow 422, 20 1269035 5 10 15 20 The two-sided turbulence 1 forms a fluid focusing effect, and the flow rate ratio of the edge sheath flow 422 to the sample fluid 2 is appropriately adjusted at this time to reduce the width of the sample fluid 2 to a width of several micrometers, that is, the sheath flow of the two sides 422 can limit the width of the sample fluid 2 to a width that allows only a single cell to pass, and the number of cells in the sample fluid 2 can be counted using a microscope (not shown). The above water fluid is deionized water, but ordinary water can be used as the water fluid as long as there is no obvious impurity in the water. However, as the water fluid composition liquid type is different, the required speed ratio of the fluid focusing will also be different. In addition, after the sample fluid 2 extruded through the edge sheath flow 422 is counted, the voltage can be applied to the electrode. Between the different electrodes of the unit 7 to drive different cells in the sample fluid to be output to the λ by different channels, for example, when the cells after counting are collected and collected into the first sorting hole 461, An alternating voltage is applied between the first electrode 71 and the first electrode 72 to generate a positive dielectrophoretic force to attract cells to the first sorting channel 46 to the first sorting hole 461 to be collected outside the base 3. Using the same principle, different types of cells can be driven to be outputted to the base 3 via the holes 461, 471 or the outflow holes 451 to facilitate recycling. In the above description, the voltage can also be negatively applied. The dielectrophoretic force classifies the cells by stimulating the output of the cells from other channels, and the implementation is not limited to the above methods. In addition, the classification channels and the main classification cells of the electrode unit 7 are set to 'not implement classification. The channel "packaged single" still has the function of cell counting, so the scope of implementation is not limited to this. Field 1 is sampled in the center of the sample stream 12 1269035 The detailed dimensions of each channel in the wafer of this embodiment, please refer to the figure. 4, wherein the width of the main nozzle 411 is 1 〇〇 micron, the width of the sub-channel 42 is 15 〇 micron, and the width of the bus channel 45 is 24 〇 micrometer, and FIG. 6 is to adjust the flow rate ratio, after the convergence channel The sample fluid 2 can be focused to a photomicrograph having a width of 5 degrees up to 17 microns in 45. Referring to Figure 7, the following is a description of the method of manufacturing the cell counting and sorting wafer of the present invention: (A) A photoresist layer 33 is coated on the top surface of the horizontal glass substrate, and is covered on the photoresist layer 33 by a photomask _1〇34 drawn with the pattern of the microchannel system 4 of FIG. The developing technique transfers the channel distribution to the substrate 31, and then etches the micro-pipe unit 4 with a glass etching solution. The micro-pipe unit has been described in detail above and will not be described again. (B) These substrates 31 are The head end of the main channel 41 is drilled with a drill to the introduction hole 412 of the bottom surface of the substrate 31, and an injection hole 441 penetrating through the bottom surface of the substrate 31 is drilled at the tip end of the water passage 1544. An outflow hole 45i penetrating to the bottom surface of the substrate 31 is drilled at the end of the bus passage 45, and a first hole is drilled at the end of the first and second sorting channels 46, 47 to penetrate the bottom surface of the substrate 31. The classification hole 461 and the second classification hole 471. The substrate is further cleaned by using a mixed solution of sulfuric acid and hydrogen peroxide for boiling for ten minutes, and then dried. (C) on the bottom surface of a glass upper plate 32 The steaming method is to lay a layer of chromium (Cr) metal and a layer of gold (Au) metal, and then develop The metal other than the electrode unit 7 is etched and etched and dried in acetone, isopropanol (Iso-Propyl AlcohoUPA), deionized water, and dried. 13 1269035 Because gold and glass adhere poorly, so use The metal interface of the chromium W is used to make the gold metal layer adhere well to the bottom surface of the upper plate. 5 10 15 20 (D) The upper plate is placed at the top surface of the substrate 3 ι and is filled with a nitrogen atmosphere. In the high-temperature sintering furnace, the heat is heated and the substrate 31 is brought into a molten state to be joined, so that the channels are hollowly formed on the substrate 3i and the upper plate 32 ι in a nitrogen atmosphere: bonding can be performed. Avoid oxidation of the metal electrode and cause wafer damage. (E) The upper plate 32 and the substrate 31 are bonded together to form a cell count sorting wafer. The cell counting classification wafer produced by the production method has the following advantages: (1) only after etching the channels, the glass substrate 31 and an upper plate 32 are spliced to each other and joined at a high temperature, & The combined structure reduces the chance of gap leakage between the glass sheets and simplifies the production process. (2) The manufacturing method can produce a cymbal sheet which can simultaneously detect a plurality of sample fluids 2, and a reticle 34 which is required to use different channel structures during the development process, without increasing the production procedure. Another object of the present invention is to provide a method for classifying wafers using the above cell counting, and referring to FIG. 8, the two sample fluids 2 in the cell counting sorting wafer are quickly captured by a digital camera 52 after the fluid is focused. The captured image is processed on a processing unit 53, and the cells are detected by image processing. The steps of the cell detection method are as follows: (A) The digital camera 52 captures a digital image of the width of the sample fluid 2 as a width that allows only a single cell to pass, and the digital camera 14 1269035 must have an orientation. The microscope head of the sample fluid 2 in the confluence channel 45 of the wafer amplifies the magnification of the photograph, enlarges the cells to a size visible to the naked eye, and the digital camera 52 outputs an image signal. (B) The image signal output by the digital camera 52 is received by a processing unit 53, and the complex image is parsed. The digital camera 52 of this embodiment takes 94 pictures per second, that is, 94 image pictures can be parsed per second by the image signal. And the captured image is a grayscale image to facilitate the discrimination of cell images. The processing unit 53 is a commonly-used computer, but the implementation is not limited to the above. (C) Selecting a region 61 in the flow of the two sample fluids 2 in the image images. (D) Monitoring the image grayscale values in the two regions 61. The image taken by the digital camera 52 is a grayscale value, and the cell particles appear blackish in the grayscale value in the image, which is quite comparable to the whiteness of the background. Using this principle, 'When the cell passes through this area 61, it will touch the sideline of the 6丨 side of the area. Because the grayscale value of the cell is darker, it is different from the background grayscale value. Change analysis, but can be identified - cell particles _ pass. (E) Counting the number of times the image grayscale value is changed in the two regions 61. The number of times the grayscale value of the two side or 61 sides of the image changes is representative of the number of cells flowing through the cell to count the number of cells in the two sample fluids 2. Referring to Fig. 9, the above method can also be used to carry out the juice number for the fine particles of different sizes. It is only necessary to circle different sizes of rectangles in the flow of each sample fluid 2 on the image in the manifold channel 45. The first 15 5 10 15 20 1269035 calls the area 63 and the enthalpy of the first and second areas 62, 63 respectively. The example is as follows: If a sample fluid containing two different sizes of cell particles is injected into the main channel In the 41st, the two cell sizes are 20 micrometers and 12 micrometers, respectively, and the width of the μ &&& field ranges 62, 63 is 20 micrometers and 12 micrometers. The size, and the two nucleus of these regions must simultaneously detect gray-scale changes in order to count - cell particles. The cells that pass through the micro-pass are passed through the first region 62 with a width of 20 microns: :: ΓΓ region 62 on both sides, so The first region 62 is not _:; Tian 肊. When the 12 micron cells pass through the second region of 12 micrometers wide (the two sides of the spectrum can simultaneously detect changes, the second region detects the number of juices - the cells pass. When a 20 micron cell appears, the first - Both sides of the second area 62, 63 can detect the change of the gray level value of the image at the same time, and the household: the two areas will _ and count - the cell passes. After the counting process, the first area 62 of the household circle is counted. The number of cells obtained is the number of true 2 μM cells, and the number of cells counted in the second region 63 counts both the number of cells of 12 μm and 20 μm, so the true number of cells of 12 μm is the second region 63 counts. The measured number minus the number of 2 () micron cells is | the true number of cells of 12 microns. It can be seen that the number of cells of two different sizes can be measured by the above method 'that is, this method can detect The number of different types of cells is also measured. The flow rate of the sample fluid 2 can also be measured by the digital image detection method. The speed measurement method is to circle the two regions 64 in the flow of the sample fluid 2 on the image in the convergence channel 45. And defining the size of the two regions 64 according to the size of the detected cells. At this time, the actual distance between the two regions 64 and the interval of 16 1269035 can be known from the image, and can be measured by the time difference of the number of movies. Time::: knowing that a cell passes the r + , B difference and distance of the two regions 64 body 2, the flow rate of the sample flow basket 2 can be calculated. The second region is 64 eves, she π nc... The cell ritibility in the actual sample fluid 2: Ε Ε , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,惶j Π time leaf number two sample fluids 2
之細胞數ϊ，並可量測彳異叉n I 、" 切不冋大小之細胞數量，及流體之 迷度。 10 15 20 歸納上述，本發明利用利用分流之觀念，僅以一系浦 就可驅動水流體於四副通道中流動形成四條均句邊鞘流， 設備構造簡單，並節省封裝、賴日㈣，更可使推動之水 流體流速平穩。其次是利用餘刻技術製作該晶片，簡化製 作程序，且避免縫隙洩漏之機會。 ^孩取後利用偵測影像灰階 值變化的方法’可同時偵測樣品流體中之不同種類之細胞 數目及樣品流體之流速。以構造簡易之震置達_測細胞 之功用，故禮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 能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飾，皆 應仍屬本發明專利涵蓋之範圍内。 【圖式簡單說明】 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 1269035 【圖式之主要元件代表符號說明】 2 樣品流體 451 流出孑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 副噴嘴 64 區域 422 邊革肖流 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 第二電極 442 分歧段 74 第四電極 45 匯流通道 19The number of cells is ϊ, and the amount of cells in the size of the divergence n I , " and the size of the fluid can be measured. 10 15 20 In summary, the present invention utilizes the concept of shunting to drive four water flows in four sub-channels to form four uniform-segmental sheath flows, with a simple structure and saves packaging, and relies on (4). It can also make the flow rate of the propelled water fluid stable. The second is to use the engraving technology to make the wafer, simplify the manufacturing process, and avoid the chance of gap leakage. ^The method of detecting changes in the grayscale value of the image after the child's can simultaneously detect the number of different types of cells in the sample fluid and the flow rate of the sample fluid. In order to achieve the function of measuring cells, it is possible to achieve the purpose of invention. The above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, that is, the simple equivalent changes and modifications made by the scope of the invention and the description of the invention are All should remain within the scope of the invention patent. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of a conventional cell counter in which the uppermost glass plate has been removed; FIG. 2 is an exploded perspective view of the conventional cell counter; 17 1269035 [The main components of the drawings represent symbolic descriptions] 2 Sample fluid 451 Outflow 孑L 3 Base 46 First sorting channel 31 Substrate 461 First sorting hole 32 Upper plate 47 Second sorting channel 33 Photoresist layer 471 Second sorting hole 34 Photomask 51 Pumping 4 Micropipe 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 leather flow 7 electrode unit 43 neck channel 71 first electrode 44 Water channel 72 second electrode 441 injection hole 73 second electrode 442 divergent segment 74 fourth electrode 45 confluence channel 19