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 ...

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.

fully-automated-centrifugal-microfluidic-device-for-ultrasensitive

Research

JoVE Journal

Bioengineering

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 vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

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 accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

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 for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

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 comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

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 neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

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 porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

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 ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Setup of a Microfluidic Device for Combinatorial Plug  Production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Author Spotlight: Integrating Computational and Experimental Approaches in Precision Oncology

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...