This work was supported by National Research Foundation of Korea (NRF) grants (2013R1A2A2A05004314, 2012R1A1A2043747), a grant from the Korean Health Technology R&#38;D Project, Ministry of Health &#38; Welfare (A121994) and IBS-R020-D1 funded by the Korean Government.

NOTE: Blood was drawn from healthy individuals and was collected in a blood collection tube. Written informed consent was obtained from all volunteers.
1. Fabrication of TiO2 NF Mat
<ol>
	<li>Preparation of precursor solution22
	<ol>
		<li>Dissolve 1.5 g of titanium tetraisopropoxide (TTIP) in a mixture of ethanol (99.9%, 3 ml) and glacial acetic acid (3 ml) and mix the solution at RT (25 &#176;C) for 30 min on a magnetic stirrer.</li>...

<ol>
	<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterials+for+Ultrasensitive+Protein+Detection.">Nanomaterials for Ultrasensitive Protein Detection.</a> Adv. Mater. 25 (28), 3802-3819 (2013).</li><li>Hu, W., Li, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterial-based+advanced+immunoassays.">Nanomaterial-based advanced immunoassays.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 3 (2), 119-133 (2011).</li><li>Yang-Kyu, C., Chang-Hoon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicon+Nanowire+Biosensor+for+Cancer+Markers.">Silicon Nanowire Biosensor for Cancer Markers.</a> Biosensors and Cancer. , 164-183 (2012).</li><li>Baltazar, R., Vistas, C. R., Ferreira, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensing+Applications+Using+Nanoparticles.">Biosensing Applications Using Nanoparticles.</a> Nanocomposite Particles for Bio-Applications. , 265-282 (2011).</li><li>Roy, P., Berger, S., Schmuki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TiO2+Nanotubes:+Synthesis+and+Applications.">TiO2 Nanotubes: Synthesis and Applications.</a> Angew. Chem. Int. Ed. 50 (13), 2904-2939 (2011).</li><li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+Nanofibrous+Membranes:+A+Novel+Solid+Substrate+for+Microfluidic+Immunoassays+for+HIV.">Electrospun Nanofibrous Membranes: A Novel Solid Substrate for Microfluidic Immunoassays for HIV.</a> Adv. Mater. 20 (24), 4770-4775 (2008).</li><li>Chantasirichot, S., Ishihara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+phospholipid+polymer+substrate+for+enhanced+performance+in+immunoassay+system.">Electrospun phospholipid polymer substrate for enhanced performance in immunoassay system.</a> Biosens. Bioelectron. 38 (1), 209-214 (2012).</li><li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+TiO2+Nanofiber-Based+Cell+Capture+Assay+for+Detecting+Circulating+Tumor+Cells+from+Colorectal+and+Gastric+Cancer+Patients.">Electrospun TiO2 Nanofiber-Based Cell Capture Assay for Detecting Circulating Tumor Cells from Colorectal and Gastric Cancer Patients.</a> Adv. Mater. 24 (20), 2756-2760 (2012).</li><li>Ali, M. A., Mondal, K., Singh, C., Dhar Malhotra, B., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-epidermal+growth+factor+receptor+conjugated+mesoporous+zinc+oxide+nanofibers+for+breast+cancer+diagnostics.">Anti-epidermal growth factor receptor conjugated mesoporous zinc oxide nanofibers for breast cancer diagnostics.</a> Nanoscale. 7 (16), 7234-7245 (2015).</li><li>Mondal, K., Ali, M. A., Agrawal, V. V., Malhotra, B. D., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Biofunctionalized+Mesoporous+Electrospun+TiO2+Nanofiber+Based+Interface+for+Biosensing.">Highly Sensitive Biofunctionalized Mesoporous Electrospun TiO2 Nanofiber Based Interface for Biosensing.</a> ACS Appl. Mater. Interfaces. 6 (4), 2516-2527 (2014).</li><li>Tu, W., Dong, Y., Lei, J., Ju, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Potential+Photoelectrochemical+Biosensing+Using+Porphyrin-Functionalized+TiO2+Nanoparticles.">Low-Potential Photoelectrochemical Biosensing Using Porphyrin-Functionalized TiO2 Nanoparticles.</a> Anal. Chem. 82 (20), 8711-8716 (2010).</li><li>Liu, S., Chen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coadsorption+of+Horseradish+Peroxidase+with+Thionine+on+TiO2+Nanotubes+for+Biosensing.">Coadsorption of Horseradish Peroxidase with Thionine on TiO2 Nanotubes for Biosensing.</a> Langmuir. 21 (18), 8409-8413 (2005).</li><li>Portan, D. V., Kroustalli, A. A., Deligianni, D. D., Papanicolaou, G. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Protein+Immobilization+Efficiency+on+a+TiO2+Surface+Modified+with+a+Hydroxyl+Functional+Group.">Enhanced Protein Immobilization Efficiency on a TiO2 Surface Modified with a Hydroxyl Functional Group.</a> Langmuir. 25 (19), 11692-11697 (2009).</li><li>Son, K. J., Ahn, S. H., Kim, J. H., Koh, W. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+Copolymer-Templated+Mesoporous+TiO2+Films+Micropatterned+with+Poly(ethylene+glycol)+Hydrogel:+Novel+Platform+for+Highly+Sensitive+Protein+Microarrays.">Graft Copolymer-Templated Mesoporous TiO2 Films Micropatterned with Poly(ethylene glycol) Hydrogel: Novel Platform for Highly Sensitive Protein Microarrays.</a> ACS Appl. Mater. Interfaces. 3 (2), 573-581 (2011).</li><li>Kar, P., Pandey, A., Greer, J. J., Shankar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh+sensitivity+assays+for+human+cardiac+troponin+I+using+TiO2+nanotube+arrays.">Ultrahigh sensitivity assays for human cardiac troponin I using TiO2 nanotube arrays.</a> Lab Chip. 12 (4), 821-828 (2012).</li><li>Agarwal, S., Wendorff, J. H., Greiner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrospinning+technique+for+biomedical+applications.">Use of electrospinning technique for biomedical applications.</a> Polymer. 49 (26), 5603-5621 (2008).</li><li>Ding, B., Wang, M., Wang, X., Yu, J., Sun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanomaterials+for+ultrasensitive+sensors.">Electrospun nanomaterials for ultrasensitive sensors.</a> Mater. Today. 13 (11), 16-27 (2010).</li><li>Liu, Y., Yang, D., Yu, T., Jiang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+electrospun+nanofibrous+PVDF+membranes+into+a+microfluidic+chip+assembled+by+PDMS+and+scotch+tape+for+immunoassays.">Incorporation of electrospun nanofibrous PVDF membranes into a microfluidic chip assembled by PDMS and scotch tape for immunoassays.</a> ELECTROPHORESIS. 30 (18), 3269-3275 (2009).</li><li>Lee, W. S., Sunkara, V., Han, J. -. R., Park, Y. -. S., Cho, Y. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+TiO2+nanofiber+integrated+lab-on-a-disc+for+ultrasensitive+protein+detection+from+whole+blood.">Electrospun TiO2 nanofiber integrated lab-on-a-disc for ultrasensitive protein detection from whole blood.</a> Lab Chip. 15 (2), 478-485 (2015).</li><li>Li, D., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Titania+Nanofibers+by+Electrospinning.">Fabrication of Titania Nanofibers by Electrospinning.</a> Nano Lett. 3 (4), 555-560 (2003).</li><li>Lombard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SolidWorks 2013 BIBLE. , (2013).</li><li>Tickoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EdgeCAM 11.0 for Manufacturers. , (2007).</li><li>Zhu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+adhesion+of+interconnected+TiO2+nanofiber+network+on+conductive+substrate+and+its+application+in+polymer+photovoltaic+devices.">Improved adhesion of interconnected TiO2 nanofiber network on conductive substrate and its application in polymer photovoltaic devices.</a> Appl. Phys. Lett. 93 (1), 013102 (2008).</li><li>Song, M. Y., Ahn, Y. R., Jo, S. M., Kim, D. Y., Ahn, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TiO2+single-crystalline+nanorod+electrode+for+quasi-solid-state+dye-sensitized+solar+cells.">TiO2 single-crystalline nanorod electrode for quasi-solid-state dye-sensitized solar cells.</a> Appl. Phys. Lett. 87 (11), 113113 (2005).</li><li>Katsuhiro, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+processed+nanofibrous+TiO2+membranes+for+photovoltaic+applications.">Electrospinning processed nanofibrous TiO2 membranes for photovoltaic applications.</a> Nanotechnology. 17 (4), 1026-1031 (2006).</li></ol>

The authors have nothing to disclose.

The assay on TiO2 NF integrated disc is a rapid, inexpensive and convenient technique for the ultrasensitive detection of low abundant proteins present in very low volume of blood. This technique has the advantage of using small sample volumes (10 &#956;l) and is amenable for analysis of multiple samples simultaneously. This provides a great potential as a multiplexing immunoassay device. The device has the added advantage that it does not require sample pretreatment steps like plasma separation, which are req...

Several platforms for disease diagnosis have been developed based on nanoscale materials1,2 such as nanowires,3 nanoparticles,4 nanotubes,5 and nanofibers (NFs)6-8. These nanomaterials offer excellent prospects in the design of new technologies for highly sensitive bioassays owing to their unique physicochemical properties. For example, mesoporous zinc oxide nanofibers have been used for the femto-molar sensitive detection of breast cancer biomarkers.9 Recently, nanomaterials based on titanium dioxide (TiO2) have been explored for bioanalytical applications10 considering their chemical stability,11 negligible protein denaturation,12 and biocompatibility.13 In addition, the hydroxyl groups on the surface of TiO2 facilitate chemical modification and the covalent attachment of biomolecules.14,15 Patterned TiO2 thin films16 or TiO2 nanotubes17 have been utilized to enhance the detection sensitivity of a target protein by increasing the surface area; however, the fabrication process is rather complex and requires expensive equipment. On the other hand, electrospun NFs are receiving attention because of their high surface area as well as straightforward and low-cost fabrication process;18,19 yet, the fragile or loose characteristic of the electrospun TiO2 NF mat makes it difficult to handle and integrate with microfluidic devices.6,20 Therefore, the TiO2 NF mats were rarely utilized in bioanalytical applications, particularly those requiring harsh washing conditions.
In this study, to overcome these limitations, we have developed a new technology for transferring the electrospun NF mats onto the surface of any target substrate by utilizing a thin polydimethylsiloxane (PDMS) adhesive layer. Furthermore, we have successfully showed the integration of electrospun TiO2 NF mats onto a centrifugal microfluidic device made of polycarbonate (PC). Using this device, a high-sensitive, fully automated, and integrated detection of C-reactive protein (CRP) as well as cardiac troponin I (cTnI) was achieved within 30 min from only 10 &#956;L of whole blood.21 Due to the combined advantages of the properties of the NFs and the centrifugal platform, the assay exhibited a wide dynamic range of six orders of magnitude from 1 pg/ml (~8 fM) to 100 ng/ml (~0.8 pM) with a lower limit of detection of 0.8 pg/ml (~6 fM) for CRP and a dynamic range from 10 pg/ml (~0.4 pM) to 100 ng/ml (~4 nM) with a detection limit of 37 pg/ml (~1.5 pM) for cTnI. These detection limits are ~300 and ~20-fold lower compared to their corresponding conventional ELISA results. This technique could be applied for the detection of any target proteins, with appropriate antibodies. Overall, this device could contribute greatly to in-vitro diagnostics and biochemical assays since it can detect rare amounts of target proteins with great sensitivity even from very small quantities of biological samples; e.g., 10 &#956;l&#160;of whole blood. Though we only demonstrated the serum protein detection using ELISA in this study, the transfer and integration technology of electrospun NFs with microfluidic devices could be more broadly applied in other biochemical reactions which require a large surface area for high detection sensitivity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Si wafer</td>
			<td>LG SILTRON</td>
			<td>Polished Wafer, test grade</td>
			<td>Dia. (mm) = 150, orientation = &#60;100&#62;, dopant = boron, RES(Ohm-cm) = 1 - 30, thickness (&#956;m) = 650 - 700</td>
		</tr>
		<tr>
			<td>Polycarbonate (PC)&#160;</td>
			<td>Daedong Plastic</td>
			<td>PCS#6900</td>
			<td>Thickness (mm) = 1 and 5&#160;</td>
		</tr>
		<tr>
			<td>Titanium tetraisopropoxide, 98%,</td>
			<td>Sigma-Aldrich</td>
			<td>205273</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone, Mw = 1,300,000</td>
			<td>Sigma-Aldrich</td>
			<td>437190</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane</td>
			<td>Sigma-Aldrich</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS and curing agent</td>
			<td>Dow Corning</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>GPDES</td>
			<td>Gelest Inc</td>
			<td>SIG5832.0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J T Baker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FE-SEM</td>
			<td>FEI</td>
			<td>Nova NanoSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray photoelectron spectroscopy</td>
			<td>ThermoFisher</td>
			<td>K-alpha</td>
			<td></td>
		</tr>
		<tr>
			<td>3D modeling machine</td>
			<td>M&#38;I CNC Lab, Korea</td>
			<td></td>
			<td>CNC milling machine</td>
		</tr>
		<tr>
			<td>Wax-dispensing machine</td>
			<td>Hanra Precision Eng. Co. Ltd., Korea</td>
			<td></td>
			<td>Customized</td>
		</tr>
		<tr>
			<td>Double-sided adhesive tape</td>
			<td>FLEXcon, USA</td>
			<td>DFM 200 clear 150 POLY H-9 V-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting plotter</td>
			<td>Graphtec Corporation, Japan</td>
			<td>Graphtec CE3000-60 MK2</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>MIDAS</td>
			<td>SPIN-3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Furnace (calcination)</td>
			<td>R. D. WEBB COMPANY</td>
			<td>WEBB 99</td>
			<td></td>
		</tr>
		<tr>
			<td>Rheometer (Tack test)</td>
			<td>Thermo Scientific</td>
			<td>Haake MARS&#160;III - ORM Package</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen plasma system</td>
			<td>FEMTO</td>
			<td>CUTE</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse antihuman hsCRP</td>
			<td>Hytest Ltd., Finland</td>
			<td>4C28</td>
			<td>(clone # C5)</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-cTnI</td>
			<td>Hytest Ltd., Finland</td>
			<td>4T21</td>
			<td>(clone # 19C7)</td>
		</tr>
		<tr>
			<td>HRP conjugated goat polyclonal anti-hsCRP</td>
			<td>Abcam plc., MA</td>
			<td>ab19175</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP conjugated mouse monoclonal anti-cTnI</td>
			<td>Abcam plc., MA</td>
			<td>ab24460</td>
			<td>(clone # 16A11)</td>
		</tr>
		<tr>
			<td>hsCRP</td>
			<td>Abcam plc., MA</td>
			<td>ab111647</td>
			<td></td>
		</tr>
		<tr>
			<td>cTnI</td>
			<td>Fitzgerald, MA</td>
			<td>30-AT43</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Amresco Inc</td>
			<td>E404</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tubes</td>
			<td>BD vacutainer</td>
			<td>367844</td>
			<td>K2 EDTA 7.2 mg plus blood 
			collection tubes</td>
		</tr>
		<tr>
			<td>SuperSignal ELISA femto</td>
			<td>Invitrogen</td>
			<td>37074</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular multilabel plate reader</td>
			<td>Perkin Elmer</td>
			<td>Envision 2104</td>
			<td></td>
		</tr>
		<tr>
			<td>Disc operating machine</td>
			<td>Hanra Precision Eng. Co. Ltd., Korea</td>
			<td></td>
			<td>Customized</td>
		</tr>
		<tr>
			<td>Photomultiplier tube (PMT)</td>
			<td>Hamamatsu Photonics</td>
			<td>H1189-210</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoCAD</td>
			<td>AutoDesk</td>
			<td>Version 2012</td>
			<td>Design software</td>
		</tr>
		<tr>
			<td>SolidWorks 3D CAD software&#160;</td>
			<td>SOLIDWORKS Corp.</td>
			<td>Version 2013</td>
			<td>3D Design software,</td>
		</tr>
		<tr>
			<td>Edgecam</td>
			<td>Vero software</td>
			<td>version 2009.01.06928</td>
			<td>Code generating software</td>
		</tr>
		<tr>
			<td>DeskCNC</td>
			<td>Carken Co.</td>
			<td>version 2.0.2.18</td>
			<td>CNC milling machine software</td>
		</tr>
	</tbody>
</table>

fully automated centrifugal microfluidic device for ultrasensitive protein detection from whole blood

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.

Using this protocol, a fully automated centrifugal microfluidic device for protein detection from whole blood with high sensitivity was prepared. The TiO2 NF mats were prepared by processes of electrospinning and calcination. In order to fabricate the NFs of desired diameter, morphology, and thickness, electrospinning conditions such as flow rate, voltage, and spinning time were optimized. When the conditions were not optimized, the quality of the NFs formed was poor. In partic...

Watch this Scientific Journal Video about Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood at JoVE.com

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L 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.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

The overall goal of this procedure is to improve the detection sensitivity of ELISA using natural spun nanofibers. This video demonstrates how to achieve femtomolar detection sensitivity of proteins in 10 microliter of whole blood samples within 30 minutes. In a lab-on-a-disc platform, the centrifugal force is used to transfer the liquid through chambers, and the total process is fully automated.The detection sensitivity could be highly enhanced by the integration of nanofibers. However, the problem was that the nanofiber is very fragile and difficult to handle. In this video, we introduce a simple process to attach fragile titanium dioxide nanofiber mats on a plastic disc.To fabricate titanium dioxide nanofiber mats on silicone substrate by electrospinning, the silicone substrate is first silanized, coated with PDMS, and precured. A nanofiber mat is then transferred to this precured PDMS and this product is punched to produce a circular piece for attachment. Coat the reaction chamber with a drop of PDMS as an adhesive, and attach the circular piece to the bottom of the chamber.Then, cure the PDMS in the oven. Finally, assemble the disc layers into one complete device for ELISA. This nanofiber transfer technique allows us to utilize the high surface area of nanofibers for bioanalysis.The centrifugal microphylitic device offers the full integration and automation of all the process required in ELISA. The main advantage of this technique over other existing methods is that it could detect femtomolar concentrations of proteins from only 10 microliter of whole blood. Prepare the precursor solutions as described in the accompanying test protocol.Then load the solution in the electrospinner and fabricate the nanofibers by electrospinning, using the optimal conditions. When the polymer nanofiber mats are prepared, calcinate them in a furnace under vacuum. After calcination, the titanium dioxide nanofiber mats are finally prepared.Prepare the microphylitic layout by using 3D designing software such as SOLIDWORKS, and transfer the design to the CNC milling machine. Operate the CNC milling machine to fabricate the disc with channels and chambers. The disc and nanofiber mat is now ready to be integrated.Prepare a vacuum chamber, silicone substrate and fluorosilane. Silanize the silicone substrate with fluorosilane under vacuum for 30 minutes. Prepare the guest PDMS by preparing prepolymer with a curing agent in a 10 to one ratio, and spin coat onto the silanized silicone substrate at 650 RPM for 60 seconds.Cure the PDMS coated on the silanized wafer at 65 degrees Celsius for 10 minutes to make it adhesive. Now transfer those nanofiber mats onto the PDMS-coated silicone by applying appropriate pressure using a flat object such as a glass slide. Bake the sample at 80 degrees Celsius for one hour to completely cure the adhesive PDMS.Dispense a few drops of ethanol on the nanofiber mats attached on the PDMS layer, and make a six millimeter hole in diameter using a PunchIt. Then peel of the punched nanofiber mats attached on the PDMS from the silicone substrate. Next, coat the binding reaction chamber in the disc with about 20 microliters of PDMS mixture, and place the nanofibers attached on the PDMS into the chambers.Then, incubate the nanofiber mat integrated disc at 80 degrees Celsius for one hour. Prepare 1%volume per volume solution of GPDES in ethanol. Then, treat the nanofibers in the disc with oxygen plasma.Dispense 100 microliters of GPDES solution on each nanofiber mat, and incubate at room temperature for two hours in a sealed container. After the incubation, remove the GPDES solution by tapping the disc on a wiper. Wash with ethanol, remove, and bake at 80 degrees Celsius for one hour.After baking, wash with ethanol and tap to remove. Repeat this process and blow dry with a nitrogen stream. For antibody immobilization, dispense diluted captured antibody solution.Keep the discs in a humidified chamber, and incubate at 37 degrees for four hours. Wash off the remaining solution with washing buffer. Now, assemble the disc using a double-sided adhesive tape.Apply reasonable pressure to assemble the layers into a complete disc. Fill the binding reaction chambers with blocking buffer and incubate at 37 degrees Celsius for one hour. Wash twice with washing buffer and store the disc at 40 degrees Celsius until use.Load the reagents, washing buffers and 10 microliter of whole blood in the reagent and sample chambers. Place the disc in a custom built visualization machine for automated immunoassay. The visualization system contains a rotor connected to a computer for automated disc operation together with a strobe light and a CCD camera for taking images.Spin the disc at 3, 600 RPM for 60 seconds to separate the red blood cells. Mix plasma with the detection antibodies. Transfer the mixture to the reaction chamber integrated with titanium dioxide nanofiber mat.After immunoreaction, wash the nanofibers by transferring the washing buffer to the reaction chamber. Then, transfer the chemiluminescent substrate solution to the binding reaction chamber, and incubate in a mixing mode for one minute. Finally, transfer the reacted substrate solution to the detection chamber.When all the reaction is finished, transfer the disc to a detection system and read the chemiluminescent signal by using custom-built software. The disc operation and the detection system can be integrated in a single platform. By optimizing voltage and the fluorate, uniform nanofiber mats were obtained as shown in this SEM image.In order to optimize the PDMS curing time, the adhesion force was measured. If it was too short, the nanofiber mat was embedded in PDMS, or peeled off if too long. After optimization, the nanofiber mat was then stably attached on the PDMS.The linear range for C-reactive protein detection from two platforms is shown in this graph. Using the nanofiber integrity lab on the disc, we were able to achieve highly enhanced detection sensitivity of C-reactive protein from only 10 microliters of serum within 30 minutes. Currently available technologies, such as 96-well plate-based ELISA require a larger sample volume and a longer detection time.Overall, nanofiber integrated disc-based ELISA showed a broad, dynamic range of six orders of magnitude with a detection limit of six femtomole of C-reactive protein. This detection sensitivity aided by high surface area provided by nanofiber integrated in the disc exceeded that of the conventional ELISA, which was limited at 2, 000 femtomoles. After watching this video, we hope you have a good understanding of how to prepare centrifugal microphylitic device, how to integrate phasal nanofibers, and how to perform onto protein detection using ELISA.This technique can be useful to detect low bonded proteins in a very small amount of biological samples.

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Research

JoVE Journal

Bioengineering

10.4K visualizações.  School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan, Republic of Korea. The overall goal of this procedure is to improve the detection sensitivity of ELISA using natural spun nanofibers. This video demonstrates how to achieve femtomolar detection sensitivity of proteins in 10 microliter of whole blood samples within 30 minutes. In a lab-on-a-disc platform, the centrifugal force is used to transfer the liquid through chambers, and the total process is fully automated.The detection sensitivity could be highly enhanced by the integration of nanofibers. Howeve...