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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 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 μL 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 μl 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.
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
2. Integration of TiO2 NF Mat into a Centrifugal Microfluidic Disc
3. Immunoassay
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...
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...
The authors have nothing to disclose.
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.
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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