TWI513510B - 離心式粒子分離暨檢測裝置 - Google Patents
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Description
本發明係關於一種粒子分離暨檢測裝置,尤其係依粒徑作為粒子分離之依據的分離裝置。
微機電系統(micro-electro-mechanical system,MEMS)是一跨領域整合技術,被視為下世代重要的核心科技,美、日、歐等先進國家均已進入此領域並積極投入研發。微機電系統技術是一種包括光、機、電、材料、控制、化學、生醫等多重科技整合的技術,利用此製造技術可使產品因微小化而提高其性能、品質、可靠度及附加價值,同時可降低製造成本。繼微電子技術帶來輕薄短小的電子系統產品後,微機電技術將對資訊、通訊、消費性電子、工業生產、生醫保健、環保工安、國防工業、農林水產、太空航空等領域造成革命性的衝擊。
微機電系統發展的觀念是由積體電路的研究開始,藉由改善傳統精密機械加工技術並應用半導體製程技術,可製作各式各樣微小元件。微機電系統製程使用光微影技術,利用光罩複製微元件在矽晶片上,可一次製造多個具有相同精密度的微元件,大量製造的功能是其製程的一大特色。
利用微機電系統技術製作的「生物晶片」深受矚目;微機電系統加工技術可把傳統生化分析中所需的微幫浦、微閥門、微過濾器、微混合器、微管道、微感測器及微反應器等元件製作在生物晶片上,以進行樣品前處理、混合、傳輸、分離和偵
測等程序,而目前的微過濾器於分離粒子或細胞的技術目前已廣泛地應用於多種領域中,在各種分離或者純化的方法中,利用微結構而將粒子或細胞依尺寸大小進行分離的方法,由於不需要額外的標定物進行辨識,且過程快速、易於操作,因此廣為使用。目前已發展了多種利用微結構而依尺寸將粒子分離之技術大致可分類為四種類型:堰型(weir-type)過濾器、柱型(pillar-type)過濾器、交叉流式(cross-flow)過濾器以及膜(membrane)過濾器。
粒子、小珠與細胞之分離有許多方面的應用,例如癌細胞偵測、血液、組織工程與化學材料純化。在生物樣本溶液中的特定細胞次群體(sub-population),以及在混合化學材料中不同大小的粒子通常需要進一步分離或純化。微結構粒子/細胞分離系統有簡單的操作步驟、對樣本不需前處理以及可與樣本分析系統(如PCR、FACS)整合等優點。故依照粒子或細胞之大小分離原理之裝置主要的優點在於不需要特定的標定物(marker)來分離粒子。而快速的粒子或細胞分離流程以及容易處理的特性在分離裝置的發展上是非常重要的。但是這種依照粒子大小分離系統中通常都存在容易阻塞的缺點,然而,阻塞會漸漸降低分離效率,故若能設計出低阻塞現象的粒子分離裝置,實為癌細胞偵測、血液、組織工程與化學材料純化等研究領域的一大助益。
為解決以粒徑分離之過濾器通常有阻塞的問題,本發明提
供一種離心式粒子分離暨檢測裝置,係配合一離心機使用,包含一底座,承接一待測物;至少一層同心圓孔壁,設置於該底座上,以區隔出複數層環形通道,該層同心圓孔壁具有複數個間距,該間距連通各層環形通道;一外壁,設置於該底座上,圍繞於該至少一層同心圓孔壁之外圍;及一上蓋,覆於該些同心圓孔壁及該外壁之上,並具有供注入該待測物之一注入口;其中該至少一層同心圓孔壁之同一層的間距相同,且內徑較小之同心圓孔壁的間距大於內徑較大之同心圓孔壁的間距。
在本發明一實施例中,該上蓋係由PDMS(polydimethylsiloxane)所製成,該外壁為圓形,且與該複數同心圓孔壁呈同心圓狀,而該離心機之離心速率為1000-4000 rpm。
在本發明另一實施例中,該離心機進一步以碰觸該離心機之轉盤使其停止之方式改變轉速,以減少阻塞現象。
經由本發明之技術特徵,可解決以粒徑大小作為分離依據之分離器普遍存在之容易阻塞的缺點,並可應用於多種領域,例如分離化學材料微粒或生物樣本,其中該生物樣本可為一癌細胞樣本、一DNA樣本或一抗原抗體樣本,當該待測物為一含有至少一目標物之生物樣本時,先與可專一性地接合於該目標物的一偶聯物粒子結合後,再注入該離心式粒子分離暨檢測裝置之注入口。
以下將配合圖式進一步說明本發明的實施方式,下述所列舉的實施例係用以闡明本發明,並非用以限定本發明之範圍,
任何熟習此技藝者,在不脫離本發明之精神和範圍內,當可做些許更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
本發明離心式粒子分離暨檢測裝置的構想如第一圖。離心式粒子分離暨檢測裝置1為一種同心圓的分離器,係配合一離心機(圖中未示)操作,離心式粒子分離暨檢測裝置1中的待測物在經由離心機的旋轉後,粒子被離心力驅動,不同粒徑的粒子經由液體流動力經過連續不同大小的孔壁13,然後相同粒徑的粒子將被侷限在同一層的環形通道121中。這個裝置應用於小尺寸粒子的分離非常有效率,例如精密化學之顆粒球或自樣本中的血液淋巴球分離。
將此構想設計為實際可用之離心式粒子分離暨檢測裝置1,請同時參考第一圖及第二圖,該圖係為本發明離心式粒子分離暨檢測裝置1之示意圖。圖中離心式粒子分離暨檢測裝置1包含一底座12,承接一待測物;至少一層同心圓孔壁13,設置於底座12上,以區隔出複數層環形通道121,該層同心圓孔壁13具有複數個間距131,該間距連通各層環形通道;一外壁14,設置於底座12上,圍繞於該至少一層同心圓孔壁之外圍;一上蓋15,覆於該些同心圓孔壁13及該外壁14之上,並具有一注入口11供注入該待測物;其中,該至少一層同心圓孔壁13之同一層的間距131相同,且內徑較小之同心圓孔壁的間距大於內徑較大之同心圓孔壁的間距。
本發明離心式粒子分離暨檢測裝置1可利用聚二甲基矽氧烷(polydimethylsiloxane,PDMS)材料製造,粒子將被PDMS結構形成的大小過濾器(filter))分離。離心機之離心速率可為1000至4000rpm,離心速率越高,樣本之分離也越快,而本發明離心式粒子分離暨檢測裝置1可將樣本分離的最低速率為1000rpm。離心時,較大的粒子會被側向流(lateral flow)洗出同心圓孔壁的間距,並於同心圓上流動而不會使同心圓孔壁的間距內阻塞,而使小粒子順暢地向外層同心圓孔壁移動,達到有效分離的效果。
以下由實施例一至實施例三,說明本發明離心式粒子分離暨檢測裝置之製造方法,並舉分離化學材料與生物樣本之實施例以供證實本發明離心式粒子分離暨檢測裝置之分離效果。
離心式粒子分離暨檢測裝置1係利用光學蝕刻技術(photolithography)以及軟式微影技術(soft-lithography)製造。所有的裝置設計皆利用AutoCAD 2004(Autodesk,San Rafael,CA)軟體,並用高解析度印表機列印CAD檔案做透明遮罩。因此,離心式粒子分離暨檢測裝置1之至少一層同心圓孔壁13、外壁14、以及上蓋15係由聚二甲基矽氧烷(PDMS)材料翻印矽模版所製成,再裝上注入口11以及覆上底座12,詳細製造方法如下:將SU-8 3050光阻劑以3000rpm的速率旋轉塗佈於矽晶圓
31之表面,再將一印有裝置負片的透明遮罩放在矽晶圓31上並以波長334 nm之紫外光曝光90秒,使曝光區域的光阻劑交聯(cross-linked)。洗去未交聯的光阻劑,使晶圓上覆有一層裝置正凸紋32,即為第三A圖之狀態。
請參考第三B圖,離心式粒子分離暨檢測裝置1經由聚二甲基矽氧烷(polydimethylsiloxane,簡稱PDMS;購於Dow Corning,USA)以及固化脫氣的彈性混合體(基底:固化劑為10:1),再對著矽翻模原版(silicon master)於80℃,經由2.5小時聚合固化以形成PDMS固化體33;其中基底為PDMS,固化劑為PDMS B劑。
請參考第三C圖,將PDMS固化體33形成的PDMS結構從矽翻模原版上剝下,個別剪下單一裝置,並用標準4 mm直徑衝壓工具(punching tools)形成注入口11以及一流出口(供分離後之樣本取出,但非必要),為第三D圖。PDMS結構經過空氣除塵(air-dusted)、二次水與乙醇多次沖洗,再置於加熱板上乾燥。玻璃底座12經過空氣除塵以及二次水與異丙醇多次沖洗後連結上PDMS結構的下方,為第三E圖。PDMS結構表面及玻璃底座12經由氧氣電漿(oxygen plasma)氧化45秒,並置於90℃加熱板上30分鐘即完成。
以一離心機旋轉離心式粒子分離暨檢測裝置1,離心力將驅使較小粒子通過PDMS結構所形成的開口與通道,而較大的粒子因其粒徑較大,將留在內圈。可經由快速改變離心機的轉速使圓圈產生側向流(lateral flow),粒子會被側流洗出開口並在同心圓附近流動而不是阻塞開口,故可以使阻塞現象降
低,此外,離心力將維持小粒徑的粒子通過開口與通道並防止它們流回較內圈的空間。
由實施例一製造一離心式粒子分離暨檢測裝置1,具有二層同心圓孔壁13,其同心圓孔壁間距之大小為內層為40 μm及外層為20 μm。
利用混合的49 μm與10 μm聚苯乙烯(polystyrene,簡稱PS)微球作為待測物,測試離心式粒子分離暨檢測裝置1之分離效果。混合的微球(請參考第四A圖)混於純水中從注入口11置入,並將離心式粒子分離暨檢測裝置1置放在一離心機(購於E-Centrifuge Wealtec Corp.)上。利用離心機以4000rpm離心20秒後,可觀察到因為孔洞尺寸以及離心力的作用,不同大小的粒子被分離。較大的49 μm聚苯乙烯微球停留在裝置的中心區域(第四B、C圖),較小的10μm聚苯乙烯微球被帶到裝置外圍區域(第四D圖)。
在實驗過程中,沒有發現明顯的阻塞現象,應是沿著同心圓的側流沖洗孔洞附近的粒子,使聚苯乙烯微球不會阻塞裝置孔洞,分離結束後,可以用注射針筒直接插入不同環形通道121中將分離後的樣本吸出,亦可如前述另開流出口將分離後的樣本取出。
利用功能性粒子與待測物結合後體積增加,此裝置將可用於生醫分子檢測。
本實施例所用之PS球購自Polysciences Inc.商品名為Polybead® Carboxylate Microspheres 20.00μm,PS球上具有COOH官能機能與蛋白質上的氨基共價鍵結。利用同為Polysciences Inc.的商品PolyLink Protein Coupling Kit,可將anti-human CD3抗體與PS球共價鍵結,其原理與鍵結方法步驟如下,並同時參考第五圖:
將微球、PolyLink結合緩衝液(Coupling Buffer)以及PolyLink沖洗/儲存緩衝液回溫至室溫,再以移液管(Pipet)吸取12.5mg之微球至一1.5-2ml的聚丙烯離心管,以500~1000 xG離心5~10分鐘,離心時間視粒徑而定,將沉澱物以0.4 ml PolyLink結合緩衝液再懸浮。
將上述再懸浮液以大約500-1000 xG再次離心5~10分鐘,其沉澱物以0.17 ml PolyLink結合緩衝液再懸浮。
使用上述微球懸浮液之前,準備200 mg/ml EDAC溶液,係由10 mg PolyLink EDAC溶於50μl PolyLink結合緩衝液中調製而成,並隨即使用。將20 μl EDAC溶液加入微球懸浮液中,以翻滾或漩渦方式輕輕攪拌使活化步驟進行15分鐘。
增加蛋白質當量至200-500 μg。如此可於結合緩衝液中作為1~5mg/ml蛋白質(LEAFTM
純化anti-human CD3抗體,購自Biolegend,Inc.)並緩慢攪拌。蛋白質結合至微球的量取決於
溶液中蛋白質的濃度以及微球的大小。
於室溫緩慢攪拌30~60分鐘,翻滾攪拌最佳,攪拌時間加長可增加蛋白質結合。
將該混合物以大約500-1000 xG離心10分鐘,取上清液評估結合蛋白質的量,並將沉澱物以0.4 ml PolyLink清洗/儲存緩衝液再懸浮,重複本段離心、再懸浮之步驟,合併上清液用於計算結合蛋白質,並將微球儲存於4℃之PolyLink清洗/儲存緩衝液中。
細胞培養部份,將人類白血病T細胞株Jurkat細胞(ATCC number TIB-152)培養於含有10%小牛血清FBS的RPMI 1640培養液(Gibco/Invitrogen)中,並於37℃,5%CO2
的培養箱環境中培養。
將表面具有anti-human CD3之PS球加入Jurkat細胞培養液中,使PS球與細胞結合,24小時後觀察其結合現象。
故依上述步驟將直徑為20 μm之Polystyrene(PS)球接上anti-human CD3抗體後,將具有Human CD3接受器之Jurkat細胞(人類白血病T細胞株Jurkat細胞:第六A圖)與PS球反應結合後(第六B圖),加入離心式粒子分離暨檢測裝置1中並置放在一離心機上以4000rpm離心,待5秒後接觸轉盤使其立刻停止,如此反覆5次或以上使分離。將具有anti-CD3之PS球及Jurkat細胞培養液之混合液體加入本發明裝置中心之液體槽中,並以小型離心機離心,所使用之離心機為E-CentrifugeWealtec Corp.,額定電壓下之離心速度約4400
RPM,相對離心力約2000g。
本發明之一較佳實施例中,分離細胞所用的離心式粒子分離暨檢測裝置1具有二層同心圓孔壁13,其同心圓孔壁13之間距131於內層為25μm,外層為15μm;同心圓孔壁13之間距131可由欲分離之樣本粒子大小決定,而環形通道121的寬度則可由樣本粒子的多寡決定,並無一定的規格。
結果顯示未與PS球結合之細胞被分離至裝置最外層(第七A圖),而與PS球結合之人類白血病T細胞株Jurkat細胞被成功留置分離至內層(第七B圖)。本實驗成功驗證此裝置結合功能性粒子後,可用於分離癌細胞或是其他特殊欲分離之細胞。以相同原理亦可分離其他檢體如:DNA及蛋白質等檢體。
經由上述實施例可知,本發明離心式粒子分離暨檢測裝置可利用標準化PDMS模組流程製造。因此擁有製造成本低、方便使用,且不同粒徑的粒子可有效被分離等優點,且解決容易阻塞之缺點。
離心式粒子分離暨檢測裝置亦可利用不同間距及環形通道大小的設計,應用不同大小的粒子之分離,亦可增加同心圓孔壁13之層數以縮小同一空間所分離的粒子的粒徑差異。可應用於血液、血球分離、癌細胞偵測、核酸雜交、組織工程、以及化學材料純化以及生化診斷方面。
1‧‧‧離心式粒子分離暨檢測裝置
11‧‧‧注入口
12‧‧‧底座
121‧‧‧環形通道
13‧‧‧同心圓孔壁
131‧‧‧間距
14‧‧‧外壁
15‧‧‧上蓋
31‧‧‧矽晶圓
32‧‧‧裝置1的正凸紋
33‧‧‧PDMS固化體
第一圖係為離心式粒子分離暨檢測裝置的構想圖。
第二圖係為離心式粒子分離暨檢測裝置之示意圖。
第三A~E圖係為離心式粒子分離暨檢測裝置之製造流程圖。
第四A~D圖係為離心式粒子分離暨檢測裝置分離聚苯乙烯微球之實驗結果。(A)混合49μm與10 μm之PS微球;(B、C)離心後,49 μm PS微球被分離由內圈孔洞分離;(D)10μm PS微球被旋轉至裝置較外層的空間。
第五圖係為anti-human CD3抗體與PS球共價鍵結之原理示意圖。
第六A圖係為人類白血病T細胞株Jurkat細胞。
第六B圖係為人類白血病T細胞株Jurkat細胞與PS球之結合狀態。
第七A~B圖係為分離細胞實驗之結果。
1‧‧‧離心式粒子分離暨檢測裝置
11‧‧‧注入口
12‧‧‧底座
13‧‧‧同心圓孔壁
131‧‧‧間距
14‧‧‧外壁
15‧‧‧上蓋
Claims (10)
- 一種離心式粒子分離暨檢測裝置,係配合一離心機使用,由下列構件組成:一底座,承接一待測物;至少二層同心圓孔壁,設置於該底座上,以區隔出複數層環形通道,該層同心圓孔壁具有複數個間距,該間距連通各層環形通道;一外壁,設置於該底座上,圍繞於該至少一層同心圓孔壁之外圍;及一上蓋,覆於該些同心圓孔壁及該外壁之上,並具有供注入該待測物之一注入口;其中,該至少一層同心圓孔壁之同一層的間距相同,且內徑較小之同心圓孔壁的間距大於內徑較大之同心圓孔壁的間距。
- 如申請專利範圍第1項所述之離心式粒子分離暨檢測裝置,其中該外壁為圓形,且與該複數同心圓孔壁呈同心圓狀。
- 如申請專利範圍第1項所述之離心式粒子分離暨檢測裝置,其中該至少一層同心圓孔壁、該外壁、以及該上蓋係由PDMS(polydimethylsiloxane)所製成。
- 如申請專利範圍第1項所述之離心式粒子分離暨檢測裝置,其中該離心機之離心速率為1000-4000rpm。
- 如申請專利範圍第4項所述之離心式粒子分離暨檢測裝置, 其中該離心機進一步以碰觸該離心機之轉盤使其停止之方式改變轉速,以減少阻塞現象。
- 如申請專利範圍第1項所述之離心式粒子分離暨檢測裝置,其中該待測物為一化學材料微粒。
- 如申請專利範圍第1項所述之離心式粒子分離暨檢測裝置,其中該待測物為一生物樣本。
- 如申請專利範圍第7項所述之離心式粒子分離暨檢測裝置,其中該生物樣本為一癌細胞樣本、一DNA樣本或一抗原抗體樣本。
- 如申請專利範圍第8項所述之離心式粒子分離暨檢測裝置,其中當該待測物為一含有至少一目標物之生物樣本時,先與可專一性地接合於該目標物的一偶聯物粒子結合後,再注入該離心式粒子分離裝置之注入口。
- 如申請專利範圍第9項所述之離心式粒子分離暨檢測裝置,其中該偶聯物粒子係為一具抗體之粒子。
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- 2012-12-05 US US13/706,021 patent/US9101943B2/en active Active
Patent Citations (5)
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TWI262815B (en) * | 2003-10-10 | 2006-10-01 | Tama Tlo Corp | Fine particles separation treatment system and cyclone-type centrifugal separation device |
TWI240652B (en) * | 2004-12-22 | 2005-10-01 | Ind Tech Res Inst | Centrifugal separation device for agitator mill |
US20070224591A1 (en) * | 2006-03-27 | 2007-09-27 | General Electric Company | Rotation-based microsampler, system and method of using the same |
TW200921102A (en) * | 2007-11-02 | 2009-05-16 | Ind Tech Res Inst | Fluid analytical device and fluid analytical method thereof |
TW201022675A (en) * | 2008-12-12 | 2010-06-16 | Univ Nat Taiwan | Compact disk based platform for separating and detecting immunomagnetic bead labeled cells |
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TW201406463A (zh) | 2014-02-16 |
US20140045249A1 (en) | 2014-02-13 |
US9101943B2 (en) | 2015-08-11 |
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