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 JoVE Biology

制备热塑性微流体通道

1, 1, 1

1Department of Biomedical Engineering, Boston University

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    Summary

    在这里我们将演示如何制造热塑性微流体芯片采用热压成型和热封。然后,我们将演示如何使用通过密封芯片执导的原位光接枝表面和聚合,形成的复合材料的固相柱。

    Date Published: 2/03/2008, Issue 12; doi: 10.3791/664

    Cite this Article

    Bhattacharyya, A., Kulinski, D., Klapperich, C. Fabrication of the Thermoplastic Microfluidic Channels. J. Vis. Exp. (12), e664, doi:10.3791/664 (2008).

    Abstract

    在我们的实验室,我们已经成功地分离出核酸,直接从人体的血液,尿液和大便使用聚合物/纳米粒子复合材料的微观溶解和固相萃取柱微升和submicroliter卷。回收的样本PCRable无需任何额外的清理,集中,小体积样品。在这里,我们将演示如何制造热塑性微流体芯片采用热压成型和热封。然后,我们将演示如何使用通过密封芯片执导的原位光接枝表面和聚合,形成的复合材料的固相柱。我们展示嫁接和碳纳米管/聚合物复合列细菌细胞裂解聚合。然后,我们显示芯片采用硅/聚合物复合材料柱从样品核酸的固相萃取的裂解过程。所附的协议包含如何使裂解和固相萃取柱的详细说明。

    Protocol

    制造微流体通道在一个循环的聚烯烃(Zeonex ® 690R,吉恩化工有限公司,路易斯维尔,肯塔基州)。 Zeonex有136℃的玻璃化转变温度(Tg)和紫外线透明的,这是光在巨石形成多孔的用于化学至关重要。微结构微热压印一个尼科电铸主模,它具有负(反)微流体平台的功能。热压印完成如下:

    1. 模具和聚合物衬底之间放置两个平行的金属压板。
    2. 该压板加热压花温度(166℃), ​​这是30 º C以上的Zeonex Tg。
    3. 的压板,然后同时按下约5分钟,并保持在250磅的压力。
    4. 模具与基板,然后取出从加热盘,让其冷却1-2分钟,然后手动相互分离。
    5. 1.5毫米直径的井钻探样品的引进和收集渠道的两端。
    6. 压纹塑料基板,然后用另一块的Zeonex热粘合,形成封闭的微流体通道。浮雕基材和覆盖件可紫外臭氧或热粘合前处理,以提高密封效率和确保封闭的渠道结构(巴塔和Klapperich,2007年)的保真度的等离子体。

    塑料微流体通道内固相萃取(SPE)柱的制备

    固相制备是一个两步的过程。起初,编造微表面改性的塑料装置,以提高固相萃取柱的附着力。通过表面聚合接枝是用于功能化的聚合物微通道的墙壁。

    嫁接过程如下:

    1. 微充满了1:1的混合物的环氧丙烯酸酯(EDA)和甲基丙烯酸甲酯(MMA)用3%的二苯甲酮,这是一个抽象的光引发剂的氢。 EDA,甲基丙烯酸甲酯和二苯甲酮均购自Sigma - Aldrich公司,圣路易斯,密苏里州。
    2. 然后,该芯片是紫外线照射,在254 nm紫外线波长和紫外线照射仪(CL - 1000紫外交联剂,UPV公司,高地,加州)200兆焦耳 /厘米2的能量为10分钟。嫁接发生拴表面的聚合物链,获得的庞然大物在第二步准备使用的聚合混合物中通过的H -抽象的聚合和结果。
    3. 从通道中删除多余的单体与15μL甲醇使用真空抽吸冲洗。

    后接枝过程中,微孔聚合​​物巨石形成的微通道内,坑害二氧化硅粒子对DNA的提取。 多孔的一块准备在原地如下:

    1. 表面改性的通道都充满了一个庞然大物的易制毒化学布玛(15%WT),EDMA(10%WT),1 - 十二醇(52.5%WT),环己醇(22.5%WT),DMPAP组成的混合物(1%WT与尊重单体)和0.7微米的二氧化硅微粒(尊重的预聚物的混合物总量的15%)。硅胶微球均购自Polysciences,公司(睡眠PA)和Sigma - Aldrich公司,圣路易斯,密苏里州购买的单体和porogenic溶剂。
    2. 该芯片是与紫外线紫外线交联剂照射在200兆焦耳/厘米2为1.1分引发聚合,从通道中删除多余的单体与15μL甲醇使用真空抽吸冲洗。
    3. (NanoPorts TM连接,厄普丘奇科学,橡树港,WA)流体连接,然后附着在微流体通道的引进和集水井。
    4. 多孔的一块,然后再与甲醇洗涤使用注射泵流量的100mL/hour至少30分钟。

    北海裂解一枝独秀制作塑料微流体通道内

    制备的CNT(碳纳米管)裂解庞然大物是一个两步的过程。首先,制造微表面改性(嫁接),在同样的方式在塑料设备的SPE柱。

    后接枝过程中,形成微孔聚合物一枝独秀内的多壁碳纳米管浸渍微。 多孔的一块准备在原地如下:

    1. 多壁碳纳米管悬浮在环己醇在2.27M的浓度。这再悬浮超声在50%幅度为30分钟和一个sonifier细胞干扰物(50%占空比Sonifier的S - 250丹伯里,必能信超声,CT)的研发,制造的。超声提供了碳纳米管的有效和稳定的0.5M的悬浮液。 Nanolabs(布赖顿,马萨诸塞州)均购自碳纳米管。
    2. 除了现在的环己醇的解决方案+使用碳纳米管是表面改性通道都充满了类似的巨石前体的混合物。一枝独秀的混合物组成的布玛(15%WT),EDMA(10%WT),1 - 十二醇(52.5%WT),环己醇+碳纳米管(22.5%WT)。此解决方案添加光引发剂DMPAP(1.13%的单体重量)。单体,porogenic溶剂和DMPAPA均购自Sigma - Aldrich公司,圣路易斯,密苏里州。
    3. 该芯片是在120兆焦耳/ 厘米 2照射紫外线紫外线交联剂为0.9分钟,引发聚合。该设备是180度翻转的crossliker,另一个在120兆焦耳/平方厘米0.9分钟照射。从通道中删除多余的单体与50μL甲醇使用真空抽吸冲洗。
    4. (NanoPorts TM连接,厄普丘奇科学,橡树港,WA)流体连接,然后附着在微流体通道的引进和集水井。

    北海裂解一枝独秀使用的细菌样品制备

    一种细菌样品的样品制备涉及到在600nm处的光密度测量,以确保该样本是不是非常集中,以免堵塞通道。

    1. 在与媒体的细菌600nm处的OD读数,这是一个分光光度计(Eppendorf公司分光光度计,科学的Eppendorf公司,韦斯特伯里,纽约州)。稀释样品,直到OD值下降的范围内0.18-0.25答:
    2. 在5分钟6500转离心样品,倒出上清液。

    裂解

    1. GuSCN悬浮细菌* + .01%SDS + 4%,蛋白酶K(0.8mg/ml)。涡简要(媒体细菌1毫升应悬浮1毫升GuSCN)。蛋白酶K被添加到解决方案援助变性,附着细菌的DNA和蛋白质抑制DNAases和RNAases。蛋白酶K的一个额外的好处是,它也打破细胞壁的蛋白质,这是可取的,因为革兰氏阳性菌和革兰氏阴性菌细胞壁中含有大量的蛋白质(虽然革兰氏阴性菌通常有更多的蛋白质艾滋病)。此外,离液剂缓冲区艾滋病的蛋白酶K与变性的蛋白质,而洗涤剂破坏细胞壁。蛋白酶K和胍硫氰酸盐(GuSCN)均购自Qiagen公司(瓦伦西亚,加利福​​尼亚州)。 SDS(​​十二烷基硫酸钠)购自皮尔斯(罗克福德,IL)。
    2. PEEK管连接到一个注射器,立即装入样品。使系统在没有空气吸入注射器。连接管nanoport和渠道。

    一个KDS100注射泵(Holliston,马KD科学,制造)用于实验和使用的流量为450μL/小时的所有步骤。流450ul/hr样品裂解通道。

    1. 收集样品,并继续通过SPE提取DNA(DNA提取协议)。注意:这种细菌样本不会需要遵循载荷步2,自暂停GuSCN *.

    DNA的提取协议

    提取过程包括3个步骤:

    1. 负载。
    2. 华盛顿州
    3. 流出。

    KDS100注射泵(Holliston,马KD科学,制造)用于实验和使用的流量为300μL/小时的所有步骤。

    负载

    1. 条件通道离液剂缓冲区(GuSCN *,硫氰酸胍)在实验开始前1-2分钟。
    2. 在离液剂缓冲液按1:1的比例混合样品。
    3. 3分钟的通道流过样品的混合物。

    1. 洗2分钟与70%乙醇的通道。
    2. 洗净与100%乙醇2分钟的渠道。

    洗脱

    1. 3分钟的微孔水洗脱提取的DNA。收集15μL纯化,DNA PCR准备。

    * GuSCN从Qiagen公司试剂盒(缓冲区RLT)。

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    Disclosures

    References

    1. Bhattacharyya, A. and Klapperich, C.M., 2007. Mechanical and chemical analysis of plasma and ultraviolet-ozone surface treatments for thermal bonding of polymeric microfluidic devices. Lab Chip 7, 876-882.

    Comments

    5 Comments

    who and where did produce the NiCo electroformed master-mold? Have you any facility for such type of devices?
    Reply

    Posted by: AnonymousMarch 27, 2008, 8:21 AM

    We do not have an in house facility for electroplating. We send out the master moulds to NiCoForm in Rochester, NY. They will take your DRIE Si or do the DRIE step for you.Catherine Klapperichcatherin@bu.edu 
    Reply

    Posted by: AnonymousMarch 27, 2008, 10:47 AM

    Here are a couple of more references that relate to this protocol: Bhattacharyya, A. and C.M. Klapperich,”Microfluidics -Based Extraction of Viral RNA for  Disposable Molecular  Diagnostics, " Sensors and Actuators B: Chemical, Volume 1²9, Issue ², ²² February ²008, Pages 693-698. Bhattacharyya, A. and C.M. Klapperich, “Design and testing of a disposable microfluidic chemiluminescent immunoassay for disease biomarkers in human serum samples,” Biomed Microdevices. ²007 Apr;9(²):²45-51. Bhattacharyya, A. and C.M. Klapperich, Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics. Anal Chem. ²006 Feb 1;78(3):788-9². 
    Reply

    Posted by: AnonymousMarch 27, 2008, 10:50 AM

    Dear Dr. Arpita Bhattacharyya
    I am working on glass microfluidic devices such as micromixer, drop generator as my PhD program. I have a question about fittings that you used in this project. How did you fit the tubings and fittings together? Would you please send me the peek tubings and fittings you have used with details?
    Yours sincerely.
    bahadori1017@yahoo.com
    Reply

    Posted by: AnonymousSeptember 7, 2009, 7:14 AM

    Great video, very informative. (I am also in the BU program)
    Reply

    Posted by: AnonymousJune 9, 2010, 12:05 PM

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