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

Summary

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

Abstract

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO₂ nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO₂ NF mat; transfer-printing of TiO₂ NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

Introduction

Several platforms for disease diagnosis have been developed based on nanoscale materials^1,2 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....

Protocol

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. Dissolve 11 wt% of polyvinyl pyrrolidone (PVP) in 3.64 g of ethanol (99.9%) and stir the solution at 65 °C for 1 hr using a hot-plate magn....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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