姜 迪，項(xiàng) 楠，唐文來，倪中華
(東南大學(xué)機(jī)械工程學(xué)院江蘇省微納生物醫(yī)療器械設(shè)計(jì)與制造重點(diǎn)實(shí)驗(yàn)室，江蘇 南京 211189)
摘要：微流控芯片的出現(xiàn)將傳統(tǒng)生化檢測帶入了微觀世界，其具有所需進(jìn)樣量少、檢測速度快、成本低廉以及便攜性高等優(yōu)點(diǎn)，實(shí)現(xiàn)了疾病的早期診斷和有效的預(yù)后評估，為提高疾病的治愈率提供了重要手段。以微操控技術(shù)為主題，結(jié)合江蘇省微納生物醫(yī)療器械設(shè)計(jì)與制造重點(diǎn)實(shí)驗(yàn)室研究成果系統(tǒng)地回顧了基礎(chǔ)的微流控芯片制造方法的發(fā)展歷程及微尺寸操控方法的技術(shù)概要，比較了幾種典型的微流控芯片的優(yōu)缺點(diǎn)及適用環(huán)境，并探討了未來微操控技術(shù)的發(fā)展方向。
關(guān)鍵詞：微流控技術(shù)；慣性流；粒子操控
1665年，英國科學(xué)家羅伯特·胡克在顯微鏡下描繪出了已經(jīng)死亡植物細(xì)胞的結(jié)構(gòu)，首次將人們的視野帶入了微觀世界。1673年，荷蘭工匠列文虎克用自制顯微鏡觀察到了血液中的紅細(xì)胞、雨水中的微生物以及動(dòng)物精子等一系列微觀的生命形態(tài)，為人類了解自身疾病的成因作出了杰出的貢獻(xiàn)。20世紀(jì)90年代初，微流控技術(shù)(microfluidics)[1](又名芯片實(shí)驗(yàn)室，lab-on-a-chip)被正式提出，開啟了大規(guī)模、精確操縱微納生物粒子的時(shí)代，為精確、快速、智能地進(jìn)行疾病檢測和監(jiān)護(hù)提供了新的思路、方法。
起初，關(guān)于微流控芯片的討論主要集中在芯片的制作方法以及材料選擇上，常采用玻璃[2]、有機(jī)硅材料[3]以及塑料[4]等進(jìn)行芯片制作。隨后，芯片的制作方法逐漸確定，各式流道也能夠方便加工，在工藝研究逐漸深入的同時(shí)微流控芯片的應(yīng)用研究也得到了長足的發(fā)展，先后出現(xiàn)了溶液的混合[5- 6]、粒子的聚焦以及分選等液體和粒子的操控技術(shù)。粒子聚焦于流道中的單一平衡位置能夠方便粒子的計(jì)數(shù)和檢測[7-8]，而微流控芯片超高的粒子分選效率能夠?qū)┌Y患者血液中數(shù)量極為稀少的循環(huán)腫瘤細(xì)胞(circulating tumor cells, CTCs)提取出來[9-12]，為癌癥患者的早期診斷和有效預(yù)后評估帶來了福音。近兩年來，隨著3D打印、智能手機(jī)以及可穿戴設(shè)備等的流行，微流控芯片的設(shè)計(jì)與制造也逐漸形成產(chǎn)業(yè)化并向智能化方向發(fā)展。譬如，一些研究團(tuán)隊(duì)已經(jīng)開始嘗試將微流控芯片和智能手機(jī)結(jié)合起來[13-14]，為人們提供隨時(shí)隨地的檢測服務(wù)并方便衛(wèi)生部門進(jìn)行數(shù)據(jù)分析。而一些新型微流控芯片制作材料如紙[15]、織物[16]以及硅膠材料的使用使微流控技術(shù)應(yīng)用于可穿戴設(shè)備成為可能[17]，這一應(yīng)用對芯片材料的親膚性以及芯片的結(jié)構(gòu)設(shè)計(jì)都提出了更高的要求。
如前所述，微流控芯片的常用材質(zhì)決定了其造價(jià)低廉，可以作為可拋式芯片，安全衛(wèi)生。微流控芯片內(nèi)微通道截面的特征尺寸較小，在幾十到幾百微米之間，所需進(jìn)樣量少，且往往芯片的通量較高，檢測速度快，能夠?qū)崿F(xiàn)樣品的快速檢測。另外，微流控芯片與智能化技術(shù)的結(jié)合使得芯片的便攜性發(fā)揮到極致，方便了患者的早期診斷且極大程度上保護(hù)了病人的隱私。微流控芯片自身的優(yōu)勢眾多，未來將是精確快速大規(guī)模微納粒子操控發(fā)展的首要載體。
本文從微流控芯片的制作方法和微納粒子的操控方法兩方面介紹微流控芯片的發(fā)展歷程，分別比較了各國學(xué)者所提出方法的優(yōu)劣及適用環(huán)境，并介紹了江蘇省微納生物醫(yī)療器械設(shè)計(jì)與制造重點(diǎn)實(shí)驗(yàn)室(以下稱本實(shí)驗(yàn)室)在芯片制作方法以及流道結(jié)構(gòu)設(shè)計(jì)方面所作出的貢獻(xiàn)。最后展望了微流控芯片的發(fā)展趨勢。
微機(jī)電系統(tǒng)(micro-electro-mechanical system)的興起為微流控芯片的加工提供了思路[18]，其主要是采用半導(dǎo)體加工工藝的光刻以及刻蝕技術(shù)將微尺度圖形轉(zhuǎn)移到基底上。由于玻璃具有透性好且易于被刻蝕等特性，故成為微流控芯片發(fā)展之初比較流行的芯片制作材料[4]。但是由于玻璃具有各向同性的特點(diǎn)，難以加工深寬比較大的流道，且玻璃材料較難鍵合，難以實(shí)現(xiàn)批量生產(chǎn)。聚合物微加工技術(shù)的出現(xiàn)使得微流控芯片的產(chǎn)業(yè)化成為可能。適合于聚合物芯片微加工工藝的常見傳統(tǒng)方法主要有熱壓、注塑、軟光刻以及適合于一些精細(xì)加工的直寫技術(shù)，近年來出現(xiàn)了一些非傳統(tǒng)的聚合物加工方法，如裂紋光刻、C-MEMS以及納米線輔助制作微納米通道等[19]，如圖1所示，為復(fù)雜圖形流道的制作提供了更為適宜的方法。
圖1 微流控芯片加工工藝的發(fā)展歷程[19]
1.1 光刻及刻蝕方法
無論是熱壓還是注塑方法，通常需要基于光刻工藝制作帶有微通道結(jié)構(gòu)的模具進(jìn)而批量生產(chǎn)聚合物芯片。一般的光刻過程主要包括在基底上旋涂光刻膠，然后在紫外曝光下通過有掩膜[20]或是無掩膜[21]的方式將流道圖案轉(zhuǎn)移到光刻膠上，顯影裸露出需要刻蝕的基底部分，通過濕法刻蝕或是干法刻蝕將流道圖案轉(zhuǎn)移到基底上，最后去掉光刻膠。濕法刻蝕即以化學(xué)溶液腐蝕基底需要刻蝕的部分，適應(yīng)性好且能夠形成均勻表面，但不適用于小尺寸圖形的刻蝕。而干法刻蝕即以等離子體刻蝕基底表面，精度較高且對環(huán)境更為友好，但制造成本較高。
1.2 壓印、熱壓及注塑方法
壓印及熱壓方法批量制作微流道的基本流程主要是：將聚合物基片與模具對準(zhǔn)并在真空中加熱聚合物至玻璃態(tài)轉(zhuǎn)化溫度以上，根據(jù)設(shè)計(jì)圖形及基片與模具的材料適當(dāng)加壓，成形后等溫冷卻至玻璃態(tài)轉(zhuǎn)化溫度以下進(jìn)行脫模[22]。模具可以用鎳鉻合金金屬線、硅片[20]以及石英[23]等硬度較高的材料制作，也可以采用電鑄方法將陰模硅片上的流道結(jié)構(gòu)轉(zhuǎn)移到金屬陽模上[24]。熱壓方法的芯片制作時(shí)間在10min左右[22]，圖形轉(zhuǎn)移效果較好，一些精度在納米級的流道結(jié)構(gòu)已經(jīng)被制作出來[25],而采用常溫下壓印的方法受材料及環(huán)境影響較大，但芯片制作時(shí)間能夠減少到2min[4]。注塑的一般步驟為將熱塑性材料加熱至融化，注入模具并進(jìn)行一段時(shí)間的加壓以補(bǔ)償材料的收縮，最后將溫度降到聚合物的玻璃態(tài)轉(zhuǎn)化溫度以下，芯片固化成形后脫模，加工時(shí)間通常為幾秒到幾分鐘[26]。注塑方法由于施加壓力較大通常需要強(qiáng)度較高的金屬材料制成模具，一般采用電鑄方法獲得鎳金屬陽模[27]，由于其擁有較好的圖形轉(zhuǎn)移效果，因此適用于微流控芯片的批量制作。
1.3 軟光刻方法
軟光刻方法即以光刻膠代替硅片及金屬模具以模塑法獲得聚合物芯片的方法[28],以聚二甲基硅氧烷(polydimethylsiloxane，PDMS)作為聚合物芯片材料最為常見。因其制作簡便、成本低、周期短、生物相容性好并且方便鍵合,軟光刻十分適合實(shí)驗(yàn)室原型器件的制作，也可以應(yīng)用于精細(xì)多層復(fù)雜流道結(jié)構(gòu)的制作[29-31]，一經(jīng)提出便得到了廣泛使用。使用軟光刻法制作聚合物芯片的一般流程為：將流道結(jié)構(gòu)經(jīng)高分辨率的激光光繪機(jī)打印在透明膠片上制得掩膜板，再在硅基底上旋涂厚度與微流道設(shè)計(jì)高度相同的SU-8負(fù)膠，在掩膜板下經(jīng)紫外線曝光后顯影得到SU-8陽模，澆注PDMS預(yù)聚物后加熱固化成形[32]。制作過程省去了金屬掩膜板及可受壓材質(zhì)陽模的制作，將微流控芯片的快速制作成本降至了最低。但是軟光刻也有一個(gè)明顯缺點(diǎn)，就是PDMS作為彈性有機(jī)硅材料在壓力較大的情況下流道截面容易變形，對粒子聚焦平衡位置產(chǎn)生影響，影響聚焦分選效果。
1.4 直寫技術(shù)
普通光刻方法的衍射效應(yīng)限制了微流道結(jié)構(gòu)的制作精度只能在微米和亞微米級，而通過應(yīng)用直寫技術(shù)可使流道精度進(jìn)一步得到提升。電子束曝光(electron beam lithography，EBL)以及聚焦粒子束(focused ion beam，F(xiàn)IB)曝光技術(shù)能夠?qū)⒆钚√卣鞒叽鐪p少至20～50nm[19]，但是加工效率低下且設(shè)備較為昂貴，一般只適用于掩膜板[33]、模具[34]以及結(jié)構(gòu)較為精細(xì)的微流控芯片[35]的原型制造。激光燒蝕技術(shù)作為一種直寫技術(shù)近些年來逐漸受到關(guān)注。應(yīng)用于微流控芯片加工的激光燒蝕技術(shù)主要包括CO2、UV和飛秒激光加工技術(shù)。CO2激光的波長為10.6μm，加工寬度范圍為100～300μm，適合加工尺寸較大的流道[36-37]。UV和飛秒激光加工技術(shù)適用于更為精細(xì)流道的加工。UV激光加工技術(shù)能夠獲得小于50μm的分辨率[38]，而飛秒激光加工技術(shù)能夠獲得更為精細(xì)復(fù)雜的3D流道結(jié)構(gòu)[39]。本實(shí)驗(yàn)室綜合成本以及精度考慮，基于UV激光加工技術(shù)，提出一套低成本、準(zhǔn)商業(yè)用途的微流控芯片加工方法[38]，如圖2所示。其基本流程為使用激光直寫技術(shù)依照微流道輪廓切割聚氯乙烯(polyvinyl chloride,PVC)薄膜，再將PVC薄膜置于上下兩層PET/EVA(polyethylene terephthalate/ethylene vinyl acetate)塑封膜中間輥壓封裝，所需制造時(shí)間小于20min且成本低廉，不僅適用于實(shí)驗(yàn)室原型器件的制作且能夠適應(yīng)工業(yè)批量生產(chǎn)的需要。
圖2 低成本激光燒蝕PVC薄膜加塑封制備芯片方法[38]
微流控技術(shù)發(fā)展到今天，產(chǎn)生了眾多的粒子聚焦以及分選方法來操縱各式微納米粒子。操控方法包括從簡單的鞘流、過濾結(jié)構(gòu)到復(fù)雜的基于外加力場，再到單純依靠水動(dòng)力方式。水動(dòng)力方式又包括單純依靠牛頓流體產(chǎn)生的慣性力以及非牛頓流體的附加黏彈性力兩種粒子作用力方式。根據(jù)粒子特性對操縱對象的區(qū)分，主要是依據(jù)聚合物粒子以及生物粒子的尺寸、柔度、形狀、介電特性、光折射率以及免疫特性等物理化學(xué)特性進(jìn)行區(qū)分。下面本文就微流控芯片各種操控方法的原理、演變以及應(yīng)用作一簡要介紹。
2.1 鞘流粒子聚焦芯片
鞘流作為出現(xiàn)較早的粒子操縱方法已經(jīng)成為粒子聚焦以及限制樣品溶液流動(dòng)區(qū)域的重要方法。根據(jù)雷諾數(shù)的定義，微米量級的流道特征尺寸使得樣品溶液在流道內(nèi)的流動(dòng)狀態(tài)基本穩(wěn)定為層流，樣品溶液能夠在鞘流夾逼作用下縮小為一條細(xì)流[7, 40]，實(shí)現(xiàn)內(nèi)部樣品粒子的聚焦。當(dāng)兩側(cè)鞘流的流量非對稱設(shè)置時(shí)，樣品溶液的聚焦流束將偏向流量小的一側(cè)，慣性作用下質(zhì)量較小的細(xì)菌將與血細(xì)胞產(chǎn)生分離[41]。以上的2D聚焦方法只能在水平或豎直方向聚焦粒子，影響計(jì)數(shù)精度。為解決該問題，3D鞘流聚焦技術(shù)應(yīng)運(yùn)而生，如采用飛秒激光加工技術(shù)得到的復(fù)雜結(jié)構(gòu)空間四鞘流粒子聚焦芯片[39]，以及由水平和豎直方向鞘流結(jié)構(gòu)復(fù)合得到的二級3D鞘流聚焦芯片等[42-46]均能較好地解決該問題。鞘流粒子聚焦芯片中樣品溶液的聚焦寬度與鞘流的進(jìn)樣量成反比，但由于樣品溶液的聚焦寬度存在極限值，為10μm左右[7, 39]，因此限制了內(nèi)部粒子的聚焦精度。
2.2 外力場粒子操控芯片
微流控芯片中粒子主要沿流體的主流方向進(jìn)行輸送，外加力場主要用來使粒子產(chǎn)生橫向遷移，進(jìn)而達(dá)到粒子聚焦以及分選的效果。經(jīng)常使用的外加力場可以籠統(tǒng)地分為聲、光、電、磁4類，這種非接觸式的粒子精確操縱方法受到了眾多研究學(xué)者的關(guān)注。
超聲波有著很強(qiáng)的粒子操控能力，粒子在超聲駐波場中會(huì)受到聲輻射壓力，粒子和承載液之間密度和可壓縮性關(guān)系的差異決定了粒子將平衡于壓力波節(jié)還是反壓力波腹處。瑞典德隆大學(xué)的NILSSON ANDREAS和FILIP PETERSSON研究小組利用此聲場粒子操控原理設(shè)計(jì)了多種微流控芯片，實(shí)現(xiàn)了包括聚焦[47]、粒子承載液交換[48]、生物粒子的分離[49]以及多尺寸粒子的排列分選[50]等多種功能。如圖3(a)所示，紅細(xì)胞聚焦于流道中心的壓力波節(jié)位置，白色的脂肪顆粒細(xì)胞聚焦于流道兩側(cè)的反壓力波腹位置。此粒子聚焦方法在細(xì)胞計(jì)數(shù)器[51-52]以及粒子夾取[53]等方面均有應(yīng)用。表面聲波(surface acoustic wave, SAW)很早便被引入了微流體操控的研究[54]，其特點(diǎn)為沿彈性物體表面?zhèn)鞑?，?shí)驗(yàn)表明其對微流體的運(yùn)動(dòng)有很明顯的影響。賓夕法尼亞州立大學(xué)的研究團(tuán)隊(duì)致力于表面聲駐波(standing surface acoustic wave, SSAW)粒子操控的研究[55-62]，其粒子聚焦原理如圖3(b)所示，叉指式換能器(interdigitated transducers，IDTs)與微流道垂直排布，聚焦粒子于流道中心，而當(dāng)IDTs與流道成傾斜排布，如圖3(c)所示，粒子及細(xì)胞能夠完成在流道截面上的跨越，可應(yīng)用于熒光標(biāo)記物的表面涂覆[63]。韓國的JEONGHUN NAM研究團(tuán)隊(duì)運(yùn)用此技術(shù)分選了不同尺寸或密度的微尺寸粒子[64-65]并實(shí)現(xiàn)了血小板于全血中的分離[66]，驗(yàn)證了SSAW對生物粒子的無損操控。
聲波是一種機(jī)械波，其傳播需要介質(zhì)，而光是一種電磁波，在真空環(huán)境下仍能傳播，發(fā)揮作用。美國貝爾實(shí)驗(yàn)室的ASHKIN于1970年提出了連續(xù)激光的光壓能夠捕獲微尺寸粒子的想法[67]；1987年，光場作為新興的粒子操縱手段，可將微尺寸粒子推動(dòng)較遠(yuǎn)的距離[68]。2003年，光場應(yīng)用于粒子連續(xù)分選微流控芯片的文章發(fā)表于Nature[69]，吸引了學(xué)者們的更多關(guān)注。其分選原理為：混合粒子在分選環(huán)節(jié)中經(jīng)棋盤格形狀的光圖案照射，其中對光勢表現(xiàn)敏感的粒子受到光壓作用產(chǎn)生橫向遷移進(jìn)而被分離出來。利用這種光鑷對微尺寸粒子的捕捉效應(yīng)，不同尺寸[70]、不同折射率[71]以及不同種類[72]的微尺寸粒子均能實(shí)現(xiàn)較好的分離，配合圖像識別技術(shù)，可以得到很高的粒子分選精度[73]。外加光場方法驅(qū)動(dòng)粒子雖然優(yōu)勢明顯但其不足之處也不容忽視，產(chǎn)生光場需要復(fù)雜且昂貴的光學(xué)設(shè)備，且由于光鑷對粒子的作用力較弱，限制了微流控芯片的通量。
圖3 聲場在微流控芯片中的應(yīng)用
介電泳是指中性物質(zhì)在非均勻電場中受到極化作用而產(chǎn)生運(yùn)動(dòng)的現(xiàn)象[74]。介電泳力的大小與粒子直徑、電導(dǎo)率和細(xì)胞內(nèi)部以及細(xì)胞膜的介電常數(shù)有關(guān)，細(xì)胞的種類、存活情況、病變情況都會(huì)影響其所受介電泳力[75]，而其力的方向與克勞修斯-摸索提因子K(ω)有關(guān)：
其中
圖4 電場在微流控器件中的應(yīng)用
磁場對鐵、鈷、鎳等物質(zhì)具有吸引作用，同樣可以運(yùn)用到生物粒子的微流控操控中。血液中的紅細(xì)胞具有天然的磁性，但瘧原蟲色素中的Fe3+表現(xiàn)出的順磁性要強(qiáng)于血紅蛋白中的Fe2+，故可以用磁場將感染瘧疾的紅細(xì)胞從健康紅細(xì)胞中分離出來[91]，如圖5所示。具有天然磁性的生物粒子在磁場中所受到的驅(qū)動(dòng)力往往較弱，而大多數(shù)的生物粒子幾乎不表現(xiàn)出磁性，使用磁場驅(qū)動(dòng)它們需要納米磁珠[92]的幫助。作為超順磁體的納米磁珠與某種生物粒子的表面抗原相對應(yīng)的特異性抗體偶聯(lián)后，若再遇到這種生物粒子，則二者發(fā)生的特異性結(jié)合反應(yīng)會(huì)使納米磁珠固定在生物粒子上。帶有這種超順磁體的生物粒子通過磁場時(shí)會(huì)受到磁場力作用進(jìn)而被分離出來，達(dá)到提取樣本的目的。應(yīng)用此種方法，可以從全血中提取白色念珠菌[93]、CTCs[94]、檢測免疫球蛋白G[95]等目標(biāo)粒子。由隸屬于強(qiáng)生公司的Veridex公司開發(fā)的CellSearch CTCs檢測系統(tǒng)就是應(yīng)用的這一原理。除了將納米磁珠和特異性抗體偶聯(lián)再與目標(biāo)粒子結(jié)合使目標(biāo)粒子表現(xiàn)出磁性的方法外，針對真核細(xì)胞，內(nèi)噬作用可以使目標(biāo)細(xì)胞將納米磁珠吞噬到細(xì)胞內(nèi)，從而達(dá)到使目標(biāo)細(xì)胞具有磁性的目的，PAMME研究小組就利用了真核細(xì)胞的這一性質(zhì)分離了小鼠的巨噬細(xì)胞和人類卵巢癌細(xì)胞(heLa cells)[96]。然而無論何種方法，納米磁珠與目標(biāo)生物粒子的結(jié)合都需要幾個(gè)小時(shí)的時(shí)間，延長了實(shí)驗(yàn)周期。除了用納米磁珠標(biāo)記目標(biāo)粒子外，鐵磁流體可以用來聚焦非磁性粒子。鐵磁流體即為納米磁珠的膠態(tài)懸濁液，可誘導(dǎo)非均勻粒子產(chǎn)生磁偶極矩，在非均勻磁場下非磁性粒子受到磁浮力的作用而聚焦在流道中心[97]。
圖5 磁場分離被瘧疾感染的紅細(xì)胞[91]
以上從聲、光、電、磁4個(gè)方面描述了微流控芯片中的外加力場。可以看到，采用外加力場方法對生物粒子進(jìn)行分選對生物粒子特征的區(qū)分比較明確，提高了生物粒子的分選精度，但是采用外加力場驅(qū)動(dòng)粒子，粒子所受到的驅(qū)動(dòng)力較弱，故而流道內(nèi)流體的流速不可過快，使粒子有足夠多的時(shí)間產(chǎn)生橫向偏移，因而該方法也限制了微流控芯片的通量。外力場操控粒子運(yùn)動(dòng)需要額外的外力場產(chǎn)生設(shè)備，芯片的制作難度和制作成本也會(huì)相應(yīng)提高，且生物粒子的磁性標(biāo)記較為耗時(shí)，限制了便攜式快速檢測微流控芯片的發(fā)展，因此一些單純依靠流道結(jié)構(gòu)、依據(jù)粒子的物理特征對其進(jìn)行分選的微流控芯片逐漸發(fā)展起來，不再需要鞘流輔助、粒子標(biāo)記以及復(fù)雜的外加力場，大大降低了微流控芯片檢測系統(tǒng)的復(fù)雜程度并簡化了檢測流程，縮短了檢測時(shí)間，為未來的疾病及時(shí)檢測系統(tǒng)提供了一個(gè)更為簡單快速的解決方案。
2.3 設(shè)障粒子分選芯片
設(shè)障粒子分選芯片采用過濾或設(shè)置障礙的方法將目標(biāo)粒子按尺寸或者是柔性特征進(jìn)行分離，可以根據(jù)設(shè)障結(jié)構(gòu)的尺寸將相應(yīng)大小的粒子分選出來[98]，柔性粒子經(jīng)過楔形過濾結(jié)構(gòu)后小尺寸粒子被隔離[99]。不對稱的分支圓柱障礙陣列也可以按尺寸分離粒子[100]，被稱作確定性側(cè)向偏移(deterministic lateral displacement，DLD)結(jié)構(gòu)，可以達(dá)到10nm的識別精度，得到了眾多學(xué)者的關(guān)注[101]，衍生出了多種關(guān)于陣列形狀的實(shí)驗(yàn)[102]、數(shù)值計(jì)算[103]以及制造工藝[104]的探討，并被應(yīng)用到了CTCs的富集[105]當(dāng)中。過濾方法操控粒子雖然操作過程較為簡單，但是有粒子發(fā)生擁堵的隱患，且設(shè)障芯片的微柱陣列結(jié)構(gòu)會(huì)給芯片制作增加一定的難度，在實(shí)際的流道結(jié)構(gòu)設(shè)計(jì)中都應(yīng)該將這些不利因素考慮進(jìn)來。
2.4 慣性流粒子操控芯片
與其他事物的發(fā)展規(guī)律相似，微流控芯片的發(fā)展也是一個(gè)由簡到繁再由繁到簡的過程。慣性流粒子聚焦芯片往往只依靠流體自身對粒子的影響對其進(jìn)行操控，不需要鞘流、外加力場以及過濾結(jié)構(gòu)的輔助就可以獲得高通量的粒子聚焦或是分選效果，降低了芯片制作難度以及外接設(shè)備的復(fù)雜性。
由前述分析可知，微尺寸流道中的絕大多數(shù)流動(dòng)為層流，故經(jīng)常省略流體的慣性效應(yīng)而將其簡化為Stokes流，而美國加州大學(xué)洛杉磯分校的慣性微流控芯片專家DINO DI CARLO指出這種簡化是有欠考慮的：“Stokes流意味著層流，但反之并不一定成立”[106]。1961年，SEGRE和SILBERBERG最早發(fā)現(xiàn)了圓管中粒子的慣性遷移現(xiàn)象，在剪切流的持續(xù)作用下，粒子將排列在流道中心和壁面之間的位置，這是Poiseuille流對粒子施加的剪切升力以及壁面施加的斥力達(dá)到平衡所產(chǎn)生的結(jié)果[107]。由此在直流道中實(shí)現(xiàn)粒子聚焦只需要對流速進(jìn)行控制，極大簡化了微流控芯片粒子操控系統(tǒng)。由于微加工工藝的原因，矩形截面流道更容易被制作且對粒子有著更好的聚焦效果，故矩形截面直流道經(jīng)常作為基礎(chǔ)的粒子聚焦結(jié)構(gòu)出現(xiàn)在慣性微流控芯片中[108-110]。正方形截面直流道能夠?qū)⒘Ｗ悠胶庥诳拷?條邊中心的4處平衡位置，而當(dāng)矩形截面的深寬比較小時(shí)，粒子由于受到豎直方向的剪切作用較為明顯，其平衡位置縮減為上下壁面中心處的2個(gè)[111]，如圖6(a)所示。當(dāng)雷諾數(shù)增大或是粒子尺寸減小時(shí)，剪切作用更為顯著，粒子平衡位置向流道外側(cè)移動(dòng)，矩形截面直流道的2處平衡位置將會(huì)再次增加至4個(gè)[112]，如圖6(b)所示。借助鞘流，高深寬比矩形截面直流道左右兩側(cè)的2個(gè)平衡位置將會(huì)縮減為1個(gè)[113]，實(shí)現(xiàn)粒子的單束聚焦。應(yīng)用高深寬比矩形截面直流道，可實(shí)現(xiàn)超高通量的生物粒子的規(guī)則排列[114]，從而可依照尺寸和柔性對粒子進(jìn)行分選[115]，將柔性較差的感染瘧疾的紅細(xì)胞和柔軟的健康紅細(xì)胞分離開來[116]，甚至可對長徑比不同的非球形生物粒子進(jìn)行區(qū)分[117-118]。
圖6 慣性直流道粒子平衡位置
東京大學(xué)的YAMADA和SEKI[119]于2005年發(fā)現(xiàn)，利用垂直于主流道的小流量支流可以將粒子規(guī)則地排列于直流道兩側(cè)并被分離出來，稱作水力過濾。具體原理為：隨著支流的不斷增加粒子也可以根據(jù)其尺寸按照由小到大的順序被分離出來，解決了以往設(shè)障結(jié)構(gòu)芯片粒子的擁堵問題。經(jīng)過后續(xù)改進(jìn)，水力過濾分選芯片的分選效率有所提高，并加入了粒子聚焦[120-121]以及分選的功能[122]。水力過濾芯片的出口較多，流道結(jié)構(gòu)較為復(fù)雜，辛辛那提大學(xué)的BHAGAT研究團(tuán)隊(duì)[123]將粒子在高深寬比矩形截面直流道中的慣性遷移現(xiàn)象引入了進(jìn)來，省略支流結(jié)構(gòu)也可將粒子排列于流道兩側(cè)并被提取出來，大大簡化了流道結(jié)構(gòu)。
單純依靠直流道對微尺寸粒子的慣性升力聚焦粒子往往需要較長距離的流道對粒子進(jìn)行持續(xù)作用，操縱效率較低，且不同尺寸粒子聚焦流束之間的距離較短，分選效果不明顯?；谝陨显颍魇綉T性微流道結(jié)構(gòu)被提出并用于在流道截面產(chǎn)生二次流，在與慣性升力的相互作用之下，不同物理特性的粒子能夠快速聚焦到區(qū)分明顯的平衡位置，提高了慣性微流控芯片的分選效率和效果。
二次流是流道橫截面的徑向流動(dòng)，因流體受到橫向擠壓而產(chǎn)生，縮擴(kuò)流道能夠有效在截面產(chǎn)生環(huán)形二次流。韓國科學(xué)技術(shù)學(xué)院(KAIST)的CHOI和PARK研究小組致力于垂直方向的斜向障礙縮擴(kuò)結(jié)構(gòu)流道的研究[124-126]。豎直方向的縮擴(kuò)結(jié)構(gòu)能夠使尺寸位于臨界值兩側(cè)的大小兩種尺寸的粒子產(chǎn)生非常明顯的分選效果，小尺寸粒子更容易被截面二次流帶動(dòng)而產(chǎn)生斜向運(yùn)動(dòng)，這一原理被應(yīng)用到了多種粒子分選芯片上[127-129]。對于橢球粒子來說，縮擴(kuò)結(jié)構(gòu)有阻礙橢球粒子在其一側(cè)聚焦的作用，應(yīng)用這一原理，可將低深寬比直流道內(nèi)上下分布的聚焦流束縮減為一個(gè)，實(shí)現(xiàn)3D聚焦[130]。水平方向的縮擴(kuò)結(jié)構(gòu)微流控芯片與豎直方向的縮擴(kuò)結(jié)構(gòu)微流控芯片相比具有制作簡單的優(yōu)勢。來自KAIST的同一團(tuán)隊(duì)于2009年提出了單側(cè)水平結(jié)構(gòu)縮擴(kuò)流道來聚焦粒子于流道的中心[131]，流道收縮段截面有Dean流產(chǎn)生，Dean流即為流道截面上下對稱的環(huán)形流動(dòng)，由流道中間層受擠壓產(chǎn)生，是二次流的一種，在Dean流的帶動(dòng)下粒子在縮擴(kuò)流道中心形成聚焦流束。韓國延世大學(xué)的研究團(tuán)隊(duì)側(cè)重于雙側(cè)縮擴(kuò)結(jié)構(gòu)流道的研究，雙側(cè)結(jié)構(gòu)的對稱性使得相同尺寸的大量粒子能夠在收縮區(qū)域聚焦在流道兩側(cè)[132]?？s擴(kuò)結(jié)構(gòu)的小尺寸空腔可導(dǎo)致周期性的壁面誘導(dǎo)力消失，小粒子更容易受流速梯度影響向流道兩側(cè)遷移而大粒子仍在流道中心位置平衡聚焦[133]，這一分選機(jī)制被多次應(yīng)用于血液中的CTCs的分離[134-136]。將雙側(cè)縮擴(kuò)流道空腔尺寸設(shè)計(jì)得較大且提高流場的雷諾數(shù)后，渦旋會(huì)占滿整個(gè)空腔，大于臨界尺寸的粒子被渦旋捕獲進(jìn)入渦旋內(nèi)部，小于臨界尺寸的粒子則仍被留在主流中[136]。有眾多研究針對漩渦對粒子的捕獲作用進(jìn)行了探討并實(shí)現(xiàn)了大尺寸粒子的提取[137-140]，為CTCs的捕捉提供了新的操縱方法。
Dean流只出現(xiàn)于單側(cè)縮擴(kuò)流道的收縮階段，而在螺旋流道當(dāng)中，處于豎直中間層的流體受離心力作用向彎曲流道外側(cè)運(yùn)動(dòng)，截面上形成了連續(xù)的Dean流漩渦，其流場分布如圖7(a)所示[141]。處于螺旋流道中的粒子在Dean流拖曳力和慣性升力的平衡下聚焦于流道內(nèi)側(cè)。當(dāng)粒子直徑足夠大使得其與流道水力直徑之比λ=d/Dh>0.07時(shí)，粒子才能在Dean流拖曳力和慣性升力的平衡下使其具有單一的聚焦位置[142-143]。不同尺寸粒子的相對聚焦位置會(huì)隨著螺旋流道的曲率δ和內(nèi)部流動(dòng)強(qiáng)度發(fā)生變化[144]。當(dāng)螺旋流道選取較大的曲率δ和較高的雷諾數(shù)Re時(shí)，粒子按照尺寸從大到小由流道內(nèi)側(cè)向外側(cè)排列[145]，這也是多數(shù)螺旋流道采取的粒子分選方式[146-147]，如對處于不同細(xì)胞周期的多尺寸細(xì)胞進(jìn)行分選[148]，對血液中的紅細(xì)胞和白細(xì)胞進(jìn)行分離[149]，這一工作方式不僅可以使粒子按尺寸有序排列并且流道可以保持很高的通量。矩形螺旋流道中尺寸相差不大的粒子可能聚焦位置較近，不利于分選，一種新的內(nèi)側(cè)壁面高度小于外側(cè)壁面高度的梯形截面螺旋流道應(yīng)運(yùn)而生[150-151]。梯形截面螺旋流道會(huì)在內(nèi)部流動(dòng)強(qiáng)度達(dá)到臨界值時(shí)粒子聚焦位置從流道內(nèi)側(cè)突然變換到靠近流道外側(cè)，且臨界流動(dòng)強(qiáng)度與粒子尺寸成正比，故流道內(nèi)的流動(dòng)強(qiáng)度介于大小粒子的臨界流動(dòng)強(qiáng)度中間時(shí)，大小粒子分別聚焦于流道兩側(cè)，提高了螺旋流道的分離精度。本實(shí)驗(yàn)室從數(shù)值計(jì)算[141]以及實(shí)驗(yàn)分析[152]兩方面對螺旋流道進(jìn)行了深入的探討，分析了不同尺寸微粒子的調(diào)控機(jī)理，并將其應(yīng)用到了人類乳腺癌細(xì)胞和血細(xì)胞的分選[153]以及血液中血細(xì)胞的分離[154]中。由于血細(xì)胞的尺寸較為分散，血細(xì)胞并沒有形成狹窄的聚焦流束，但仍能夠基本聚集在流道內(nèi)半側(cè)，實(shí)現(xiàn)血液中血細(xì)胞的提取，如圖7(b)所示。由于螺旋慣性流道中粒子聚焦的流速敏感性，為保障粒子分選效果，本實(shí)驗(yàn)室制作了一種新型五層結(jié)構(gòu)流量閥用于保障粒子的分選效果，如圖8(a)所示[155]。在此基礎(chǔ)上，巧妙地將流量閥作為氣體阻尼器用于穩(wěn)定氣源的壓力波動(dòng)，使得低成本氣動(dòng)進(jìn)樣成為可能，如圖8(b)所示[156]。
圖7 慣性螺旋流道
圖8 被動(dòng)流量閥穩(wěn)定慣性分選芯片進(jìn)樣流速
由于彎流道中Dean流上下對稱分布，微尺寸粒子能夠平衡于流道的中心層，加之鞘流的輔助則粒子能夠?qū)崿F(xiàn)3D聚焦[46, 157-158]。2007年，DI CARLO提出了非對稱蛇形慣性流道，不對稱的連續(xù)彎流道結(jié)構(gòu)同樣能在流道截面產(chǎn)生唯一的粒子聚焦位置，省去了鞘流帶來的繁瑣[159]。若與大深寬比慣性直流道結(jié)合則能夠?qū)崿F(xiàn)高聚集度的3D單束聚焦[160]。非對稱蛇形流道一經(jīng)提出便被應(yīng)用到了粒子檢測芯片的前處理步驟中[161]。本實(shí)驗(yàn)室在聚焦紅細(xì)胞的實(shí)驗(yàn)中發(fā)現(xiàn)[162]，利用非對稱蛇形流道聚焦紅細(xì)胞，無論圓盤形的紅細(xì)胞在流道中處于怎樣的姿態(tài)，紅細(xì)胞都能保持在較窄的聚焦流束內(nèi)，實(shí)現(xiàn)較為理想的聚焦效果。與尺寸相近的聚苯乙烯小球粒子相比，紅細(xì)胞的聚焦位置更靠近流道外側(cè)，如圖9(a)所示，而這與紅細(xì)胞的柔性有關(guān)。非對稱蛇形流道擁有優(yōu)秀的聚焦效果、較低的工作條件要求以及易于集成的特點(diǎn)使其擁有廣泛的應(yīng)用前景，但其對不同尺寸粒子的區(qū)分效果并不理想且小尺寸粒子較難聚焦[163]。與非對稱蛇形流道相比，對稱蛇形流道內(nèi)部粒子的聚焦形式隨流動(dòng)強(qiáng)度的演化更為復(fù)雜[164]，與小粒子相比，大尺寸粒子更傾向于聚焦于流道中心。澳大利亞臥龍崗大學(xué)的張俊提出了一種方波形狀的對稱蛇形流道結(jié)構(gòu)[165-166]，對于不同尺寸粒子的分離有較好的效果。本實(shí)驗(yàn)室對蛇形流道內(nèi)粒子聚焦過程進(jìn)行了數(shù)值計(jì)算，發(fā)現(xiàn)了Dean流漩渦可以使大尺寸粒子產(chǎn)生截面上的旋轉(zhuǎn)并聚焦于流道中心附近，相比于小粒子有著更穩(wěn)定的聚焦效果[167]。且通過豎直方向的流場分布可以得到粒子的聚焦軌跡分布受Dean流的影響非常明顯，如圖9(b)所示。
圖9 蛇形流道內(nèi)粒子的聚焦特性
2.5 慣性-黏彈性流聚焦芯片
在SEGRE和SILBERBERG發(fā)現(xiàn)粒子的慣性遷移現(xiàn)象后不久，1963年，麥吉爾大學(xué)的研究學(xué)者便發(fā)現(xiàn)了圓管內(nèi)非牛頓流體低雷諾數(shù)Poiseuille流中粒子聚焦于流道中心的現(xiàn)象[168]。2007年LESHANSKY第一次將這一原理應(yīng)用到了低深寬比的矩形截面微流道中[169]。韓國亞洲大學(xué)的研究小組將黏彈性和慣性流的性質(zhì)結(jié)合起來，實(shí)現(xiàn)了正方形截面微流道的單一平衡位置聚焦[170]，如圖10(a)所示。雷諾數(shù)Re表示流體慣性作用的強(qiáng)弱，黏彈性作用的強(qiáng)弱由韋森伯?dāng)?shù)Wi表示。黏彈性流中粒子有向剪切率低的方向遷移的特性，故粒子聚焦于流道截面中心以及4個(gè)角落位置，與慣性流耦合后，角落平衡位置不再穩(wěn)定，故能夠聚焦于流道中心。其后該研究小組又將黏彈性流體應(yīng)用于DNA分子的聚焦中[171-172]，由于DNA分子具有可變形的特性，會(huì)受到較強(qiáng)的壁面排斥力，故DNA分子只會(huì)聚焦于流道中心。D′AVINO小組探明了圓管中的黏彈性流體同樣能夠?qū)⒘Ｗ泳劢褂诩羟新瘦^低的截面中心以及靠近流道壁面的環(huán)形位置，且根據(jù)粒子的初始位置以0.8倍管道半徑為界限進(jìn)行劃分[173]。韓國高麗大學(xué)的NAM研究小組同樣對黏彈性流體中粒子的遷移有著較深入的研究，較
圖10 黏彈性流體輔助聚焦
早利用了大尺寸粒子在黏彈性流體中向流道中心聚焦速度快的原理結(jié)合鞘流實(shí)現(xiàn)了大小粒子的分離[174]，并多有應(yīng)用[175-176]。其后該小組針對黏彈性流承載液種類的差異對不同尺寸粒子的聚焦影響也做了相關(guān)的研究[177]。中國科學(xué)院力學(xué)研究所的劉超提出，雖然黏彈性流體中大尺寸粒子向流道中心的聚焦速度較快，但是如果微流道足夠長的話粒子最終的平衡位置結(jié)果是小粒子聚焦于流道中心，而大粒子向流道壁面偏移，這與大粒子對周圍流場的擾動(dòng)較為劇烈有關(guān)[178]。隨后NAM更進(jìn)一步，利用二級分選擴(kuò)大了大小粒子之間的聚焦距離[179]，在第一級分選中兩種尺寸粒子聚焦于流道中心，且大粒子向壁面偏移，二級分選中二者在流體彈性力的作用下聚焦流束之間距離加大。
近些年黏彈性流體被應(yīng)用到了更復(fù)雜的領(lǐng)域，美國克萊姆森大學(xué)的LIU發(fā)現(xiàn)黏彈性流體能夠提升慣性流對相似形狀粒子的區(qū)分程度，并成功將球形粒子和與其形狀相接近的花生形粒子區(qū)分開來[181]。澳大利亞臥龍崗大學(xué)的YUAN將黏彈性流體引入到單側(cè)縮擴(kuò)流道中，發(fā)現(xiàn)在Dean流拖曳力、慣性升力以及黏彈性作用力共同作用下粒子能夠更好地聚焦于縮擴(kuò)流道內(nèi)遠(yuǎn)離空腔的一側(cè)[182]，并應(yīng)用此原理成功提取了血液中的血細(xì)胞[183]。本實(shí)驗(yàn)室對圓截面[184]和方截面[185]直流道內(nèi)黏彈性流體對微尺寸粒子遷移行為的調(diào)控機(jī)理做了更深入的研究，并將黏彈性流體引入了Dean流連續(xù)作用的螺旋流道中[180]，發(fā)現(xiàn)在特定的流道截面深寬比以及特定的流速控制條件下粒子能夠?qū)崿F(xiàn)螺旋流道外側(cè)的單束聚焦，如圖10(b)所示，螺旋流道中StageⅤ代表粒子能夠單束聚焦，當(dāng)深寬比AR為1/4和1/2以及流道半徑Ri=1.1mm時(shí)，出現(xiàn)粒子單束聚焦的現(xiàn)象。黏彈性流體的使用會(huì)一定程度上影響慣性流的通量，但黏彈性流體的適當(dāng)應(yīng)用可以優(yōu)化粒子慣性聚焦的效果。
本文簡要介紹了微流控芯片中微納尺寸操控技術(shù)及其所依托的微流控芯片加工方法。在微流控芯片的加工方法中,壓印、熱壓以及注塑方法比較適合微流控芯片的批量生產(chǎn)，而光刻和直寫技術(shù)相對適合實(shí)驗(yàn)室原型器件、掩膜板以及模具的制造。在微流控芯片的設(shè)計(jì)中，外加力場方法對粒子的區(qū)分精度更高，但是慣性流有著超高的粒子處理效率和極低的成本需求，應(yīng)針對不同檢測粒子對象的目標(biāo)特性合理設(shè)計(jì)微流控芯片。
未來的微流控芯片將會(huì)在操控精度和效率上實(shí)現(xiàn)兼顧，對粒子的柔性和形狀等復(fù)雜特性建立更為成熟的操控分選方法。微流控設(shè)備逐漸向產(chǎn)業(yè)化和智能化的方向發(fā)展，獲得高精度和高效率的微流控設(shè)備可以通過優(yōu)化流道結(jié)構(gòu)以及設(shè)計(jì)多級分選芯片等方法來實(shí)現(xiàn)。微流控芯片的制作材料、操控方法以及驅(qū)動(dòng)方式需要向能夠與可移動(dòng)設(shè)備相匹配的方向發(fā)展。隨著智能化設(shè)備發(fā)展的不斷深入，人們自身健康意識的不斷加強(qiáng)，微流控芯片未來將會(huì)散發(fā)出更加蓬勃的生機(jī)與活力。
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JIANG Di, XIANG Nan, TANG Wenlai, NI Zhonghua
(Jiangsu Key Laboratory for Design and Manufacture of Micro-Nano Biomedical Instruments, Southeast University, Jiangsu Nanjing, 211189, China)
Abstract：The advent of microfluidic chips brings laboratory tests to the micro world and simplifies the early diagnosis for patients, helps to detect and treat the disease as early as possible and improves the chance of healing. This paper reviews the fabrication and development of the microfluidic chips, compares several typical kinds of microfluidic chips and their appropriate applications. Finally, it summaries microfluidics researches of our laboratory and the future of microfluidic chips.
Key words：microfluidic technology; inertial flow; particle manipulation
DOI：10.3969/j.issn.2095-509X.2017.03.002
收稿日期：2017-01-12
基金項(xiàng)目：國家自然科學(xué)基金資助項(xiàng)目(51505082，51375089)；江蘇省自然科學(xué)基金資助項(xiàng)目(BK20150606)
作者簡介：姜迪(1987—)，女，吉林長春人，東南大學(xué)博士研究生，主要研究方向?yàn)槲⒓{醫(yī)療器械設(shè)計(jì)與制造。
中圖分類號：TH789
文獻(xiàn)標(biāo)識碼：A
文章編號：2095-509X(2017)03-0009-14
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