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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

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

Published: April 16th, 2016

DOI:

10.3791/54143

1Department of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan, Republic of Korea, 2Agency for Defense Development (ADD), Daejeon, Republic of Korea, 3KOGAS (Korea Gas Corporation) Research Institute, 4Center for Soft and Living Matter, Institute for Basic Science (IBS)

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 µL of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

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.

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 chem....

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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

  1. Preparation of precursor solution22
    1. 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 °C) for 30 min on a magnetic stirrer.
    2. .......

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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.......

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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 μl) 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.......

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

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

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