全自动离心微流体装置从全血超灵敏蛋白检测
Summary
This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 µL of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.
Abstract
Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.
Introduction
基于纳米材料的1,2-如纳米线，3纳米颗粒，4纳米管，5和纳米纤维（NFS）6-8疾病诊断多种平台已经制定出来。这些纳米材料提供新技术的设计，由于其独特的物理化学性质高度敏感的生物测定极好的前景。例如，氧化物中孔锌纳米纤维已经被用于乳腺癌的生物标志物的毫微微摩尔灵敏的检测。9最近，基于二氧化钛（TiO 2）已探索生物分析应用10考虑到它们的化学稳定性的纳米材料，11微不足道蛋白质变性，12和生物相容性。13另外， 二氧化钛的表面上的羟基基团促进 化学修饰和生物分子的共价连接。14,15图案化的 TiO 2 THIN薄膜16或TiO 2纳米管17已被用于通过增加表面积以增强靶蛋白的检测灵敏度;然而，制造工艺是相当复杂的，并且需要昂贵的设备。另一方面，静电会员协会受到关注，因为它们的高表面积以及简单和低成本的制造过程; 18,19然而，该静电的 TiO 2的NF垫的易碎或松散特性使得它难以处理和与微流体装置因此集成。6,20，在TiO 2的NF垫中的生物分析应用很少使用，特别是那些需要苛刻的洗涤条件。
在这项研究中，要克服这些限制，我们已经开发出一种新技术，用于通过利用一个薄聚二甲基硅氧烷（PDMS）粘合剂层的静电NF垫转印到任何目标衬底的表面上。 Furthermore，我们已经成功地显示出静电的 TiO 2的NF垫的整合到由聚碳酸酯（PC）的离心微流体装置。使用这种装置，高敏感的，完全自动化，并集成检测C-反应蛋白（CRP）以及心肌肌钙蛋白I（cTnI水平）溶液在30分钟内从仅10微升的全血21来实现的。由于该组合NFS和离心平台的性能优势，该测定显示出六个数量级的宽动态范围从1微克/毫升（〜8 FM）到100纳克/毫升（〜下午12点08分）与检测的下限0.8微克/毫升（〜6 FM）为CRP和100纳克/毫升（〜4纳米）为37微克/毫升（〜下午1点05分）的检测限的动态范围从10微克/毫升（~0.4 PM） cTnI浓度。这些检测限〜300和〜20倍下相比，他们的相应的常规ELISA结果。这种技术可应用于用于检测任何靶蛋白，用适当的抗体。总体来说，这款设备合作ULD大大有助于在体外诊断和生物化学试验，因为它可以从非常少量的生物样品甚至检测罕见量的靶蛋白具有高度的敏感性; 例如 ，10微升的全血。尽管我们仅展示在这项研究中使用ELISA血清蛋白的检测，静电会员协会与微流体设备的传输和集成技术，可以更广泛地用于需要高检测灵敏度大表面积其他生化反应的应用。
Protocol
Representative Results
Discussion
TiO 2的NF集成光盘上的检测是存在于血液量非常低低丰度蛋白进行超灵敏检测快速，廉价和方便的技术。该技术具有使用小样品体积（10微升）的优点，并且是适合用于同时多个样品的分析。这提供了巨大的潜力作为一个复用免疫测定装置。该装置具有附加的优点，它不需要像血浆分离，这需要在常规的ELISA样品预处理步骤。此外，该设备可以执行整个免疫测定程序自动地（ 例如 ，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由韩国国家研究基金会（NRF）赠款（2013R1A2A2A05004314，2012R1A1A2043747），来自韩国的健康科技研发项目，卫生部和福利（A121994）和IBS-R020-D1的授予由韩国政府资助的支持。
Materials
| Si wafer | LG SILTRON | Polished Wafer, test grade | Dia. (mm) = 150, orientation = <100>, dopant = boron, RES(Ohm-cm) = 1 – 30, thickness (μm) = 650 – 700 |
| Polycarbonate (PC) | Daedong Plastic | PCS#6900 | Thickness (mm) = 1 and 5 |
| Titanium tetraisopropoxide, 98%, | Sigma-Aldrich | 205273 | |
| Polyvinylpyrrolidone, Mw = 1,300,000 | Sigma-Aldrich | 437190 | |
| Acetic acid | Sigma-Aldrich | 320099 | |
| Anhydrous ethanol | Sigma-Aldrich | 459836 | |
| Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane | Sigma-Aldrich | 448931 | |
| PDMS and curing agent | Dow Corning | SYLGARD 184 | |
| GPDES | Gelest Inc | SIG5832.0 | |
| Ethanol | J T Baker | ||
| FE-SEM | FEI | Nova NanoSEM | |
| X-ray photoelectron spectroscopy | ThermoFisher | K-alpha | |
| 3D modeling machine | M&I CNC Lab, Korea | CNC milling machine | |
| Wax-dispensing machine | Hanra Precision Eng. Co. Ltd., Korea | Customized | |
| Double-sided adhesive tape | FLEXcon, USA | DFM 200 clear 150 POLY H-9 V-95 | |
| Cutting plotter | Graphtec Corporation, Japan | Graphtec CE3000-60 MK2 | |
| Spin coater | MIDAS | SPIN-3000D | |
| Furnace (calcination) | R. D. WEBB COMPANY | WEBB 99 | |
| Rheometer (Tack test) | Thermo Scientific | Haake MARS III – ORM Package | |
| Oxygen plasma system | FEMTO | CUTE | |
| Monoclonal mouse antihuman hsCRP | Hytest Ltd., Finland | 4C28 | (clone # C5) |
| Monoclonal mouse anti-cTnI | Hytest Ltd., Finland | 4T21 | (clone # 19C7) |
| HRP conjugated goat polyclonal anti-hsCRP | Abcam plc., MA | ab19175 | |
| HRP conjugated mouse monoclonal anti-cTnI | Abcam plc., MA | ab24460 | (clone # 16A11) |
| hsCRP | Abcam plc., MA | ab111647 | |
| cTnI | Fitzgerald, MA | 30-AT43 | |
| Bovine Albumin | Sigma-Aldrich | A7906 | |
| PBS | Amresco Inc | E404 | |
| Blood collection tubes | BD vacutainer | 367844 | K2 EDTA 7.2 mg plus blood collection tubes |
| SuperSignal ELISA femto | Invitrogen | 37074 | |
| Modular multilabel plate reader | Perkin Elmer | Envision 2104 | |
| Disc operating machine | Hanra Precision Eng. Co. Ltd., Korea | Customized | |
| Photomultiplier tube (PMT) | Hamamatsu Photonics | H1189-210 | |
| AutoCAD | AutoDesk | Version 2012 | Design software |
| SolidWorks 3D CAD software | SOLIDWORKS Corp. | Version 2013 | 3D Design software, |
| Edgecam | Vero software | version 2009.01.06928 | Code generating software |
| DeskCNC | Carken Co. | version 2.0.2.18 | CNC milling machine software |
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