Name: fully automated centrifugal microfluidic device for ultrasensitive protein detection from whole blood
Uploaded: 2016-04-16T15:00:00
Description: Watch this Scientific Journal Video about Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood at JoVE.com

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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+biocompatibility+between+TiO2+nanotubes+layer+and+human+osteoblasts.">On the biocompatibility between TiO2 nanotubes layer and human osteoblasts.</a> J.Biomed.Mater.Res. Part A. 100 (10), 2546-2553 (2012).</li><li>Dettin, M., 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=Covalent+surface+modification+of+titanium+oxide+with+different+adhesive+peptides:+Surface+characterization+and+osteoblast-like+cell+adhesion.">Covalent surface modification of titanium oxide with different adhesive peptides: Surface characterization and osteoblast-like cell adhesion.</a> J. Biomed. Mater. Res. Part A. 90 (1), 35-45 (2009).</li><li>Kim, W. -. 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 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 ...

Research

JoVE Journal

Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...