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 particular, NFs were not spun out from the polymer solution at low voltage (5 kV), and were broken when the voltage was high (>20 kV). Similarly, spinning time was critical for the fabrication of NFs with appropriate density, not too sparse (1 min) or too dense (30 min). Also, to avoid bead formation caused by high flow rates, the flow rate was manipulated. So, an applied voltage of 15 kV, 10 min electrospinning time and a flow rate of 0.3 ml hr-1 were used as optimal conditions for NF fabrication based on the morphology and thickness of the resulting NFs (Figure 1).
The TiO2 NF mat was successfully transferred onto the target substrate using an adhesive PDMS layer, which was prepared by spin-coating PDMS on a silanized silicon substrate and pre-curing it in an oven at 65 °C. The time for pre-curing the PDMS layer was optimized by checking the adhesion force, using a tack test (Figure 2A). Figure 2B shows the effect of curing time on nanofiber attachment. If the PDMS was heated for 3 min, there is no adhesion force as the PDMS was still uncured and remains as a liquid and the NFs got embedded into the PDMS layer. When it is heated for 30 min, the adhesion force is very weak as the PDMS becomes stiff and loses its sticky property and the NFs were not attached on it. When the PDMS was heated for 10 min, the PDMS showed strong adhesion force and the NFs were attached strongly. From the results, it is concluded that the optimal time for pre-curing PDMS for strong attachment is 10 min.
After disc assembly, the immunoassay was conducted on the TiO2 NF mat integrated disc by following a series of disc operation processes as listed in Table 1. For the determination of CRP and cTnI concentrations in unknown samples, calibration graphs for each were made by plotting the relative light units (RLU) versus the CRP or cTnI concentration (Figure 3). In Figure 3A and 3B, the on-disc assay results were compared with conventional ELISA on a 96-well plate for CRP and cTnI respectively. The assays exhibited a broad linear dynamic range with a detection limit of 0.8 pg/ml (~6 fM) for CRP and 37 pg/ml (1.5 pM) for cTnI on a disc, which is about 300 times higher for CRP and 20 times higher for cTnI compared to their respective conventional ELISA results (286 pg/ml, 2.3 pM for CRP; 824 pg/ml, 32 pM for cTnI).
Figure 1. Optimization of electrospinning conditions. SEM images at each condition show the morphology of NFs formed. Please click here to view a larger version of this figure.
Figure 2. Tack test of adhesive PDMS layer and SEM images of the transferred TiO2 NF mat. (A) Adhesion force of the adhesive PDMS layer, pre-cured for several lengths of time, (B) SEM images of nanofibers attached on PDMS cured for different lengths of time. This figure has been modified from Ref. 21. Please click here to view a larger version of this figure.
Figure 3. Calibration graphs for the detection of CRP and cTnI using lab-on-a-disc and 96-well plate. Detection of CRP spiked in CRP-free serum (A) and cTnI spiked in whole blood (B) were conducted. The error bars indicate the standard deviation of at least three independent measurements. The limit of detection (LOD) was calculated by 3 times of the standard deviation of the negative control data measured with CRP-free serum or whole blood without cTnI spiking for CRP and cTnI, respectively. Please click here to view a larger version of this figure.
Spin No. | Speed (rpm) | Valve No. | Time (sec) | Operation | |||
1 | 3,600 | 60 | blood separation | ||||
2 | 2,400 | 1 | 3 | open valve to transfer the plasma into the chamber containing 8 µl of detecting antibodies conjugated with HRP | |||
3 | 15 Hz, 15° | 5 | mix plasma and detecting antibodies | ||||
4 | 2,400 | 2 | 3 | open valve to transfer the mixture into binding reaction chamber | |||
5 | 60 Hz, 2° | 1,200 | mix capture antibodies on TiO2 NF mat and plasma-detecting antibodies mixture | ||||
6 | 2,400 | 3 | 10 | open valve to remove the mixture | |||
7 | 2,400 | 4 | 4 | open valve to transfer washing buffer | |||
8 | 30 Hz, 30° | 120 | mix washing buffer and wash TiO2 NF mat | ||||
9 | 2,400 | 4 | transfer washing buffer | ||||
10 | 30 Hz, 30° | 120 | mix washing buffer and wash TiO2 NF mat | ||||
11 | 2,400 | 4 | transfer washing buffer | ||||
12 | 30 Hz, 30° | 120 | mix washing buffer and wash TiO2 NF mat | ||||
13 | 2,400 | 4 | transfer washing buffer | ||||
14 | 30 Hz, 30° | 120 | mix washing buffer and wash TiO2 NF mat | ||||
15 | 2,400 | 20 | remove the remaining washing buffer | ||||
16 | 5 | close valve | |||||
17 | 2,400 | 6 | 10 | open valve and transfer the chemiluminescent substrate | |||
18 | 30 Hz, 2° | 60 | mix substrate and the immunoreagents on TiO2 NF mat | ||||
19 | 2,400 | 7 | 10 | open valve and transfer the reacted substrate to the detection chamber | |||
Total time | ~ 30 min |
Table 1. Operation program for the immunoassay in a disc. This table has been modified from Ref. 21.
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 required in conventional ELISAs. Moreover, the device can perform whole immunoassay procedures (e.g., mixing, washing, binding etc.) automatically due to pre-designed chambers, channels, and valves. Additionally, using commercialized disc fabrication methods (e.g., injection molding, UV/ultrasonic/thermal bonding) instead of milling and manual assembly using adhesives, the whole procedure from device fabrication to analysis can be automated.
Also, the transfer-printing method presented here shows a simple and easy transfer of electrospun NF from donor to target substrate by utilizing a thin adhesive PDMS layer. In general, the TiO2 NFs obtained after the calcination process are brittle and easily peel off from the grounded substrate due to weak adhesion.25 Due to its characteristic brittleness, integrating the TiO2 NF mats with other devices is difficult. To solve this, many methods have been reported. For example, hot-press26 and solvent-vapor27 techniques showed the enhancement of the adhesion between TiO2 NFs mat and target surface before calcination process. Even though these methods increased the adhesion, the TiO2 NF mats were not intact due to the high mechanical pressure or the solvent applied during the respective techniques. Furthermore, because of the calcination step, integration of the TiO2 NF mats using these methods can be applied only for limited target surfaces.
To the best of our knowledge, this is the first technique to show the transfer-printing of NF mats on to a device made of thermoplastics, retaining the novel properties of NFs even after the transfer. For better performance of the device, fabrication of high-quality NFs and optimization of pre-curing conditions are desired. As mentioned in the protocol, the time required for pre-curing may vary depending on the environmental conditions; therefore, pre-curing conditions are to be optimized by measuring the adhesion force using a tack-test.
Furthermore, the protocol presented here, provides skillful guides for the fully automated ELISA process on a disc. The protocol introduces not only fabrication of the disc but also the automation of all the processes required for ELISA, including separation of whole blood, metering, mixing, washing and detection. Each step is optimized with spin speed, mixing frequency, and operation time. Based on this operation guide, the reagents loaded on a disc can be transferred utilizing a single motor. Since the device performed excellent detection sensitivity with very low volume of blood, it provides a great potential for diagnostic applications by screening biomarkers at an early stage of disease. The device would be enabled to detect any biomarker depending on the availability of its specific antibodies.
Although the device with TiO2 NF mats achieved highly enhanced sensitivity due to the large surface area of the mats, one of the remaining challenge of this technique could be in the mass production of uniform NF mats. Because the sensitivity of the ELISA is strongly affected by the surface area of the nanofiber mat, it is critical to achieve not only a high surface area but also a reproducible and uniform surface area in different batches of mass production.
In conclusion, we have shown a simple method to integrate a brittle NF mat into a functional device for ultrasensitive protein detection. The advantages of this method are as follows: 1) previously electrospun TiO2 NFs could be prepared only on conductive and thermally stable surfaces; here, we have shown that they can be transferred onto any substrate including non-conductive and plastic materials; 2) the TiO2 NF mats can withstand pressure and remain stable during washing steps due to the high adhesion force of PDMS layer; 3) it provides a relatively higher surface area for bioassays; and 4) the antibodies can be covalently attached to the TiO2 NFs due to the intact surface properties on NFs. This technique has great potential to be used for integration of nanofibrous materials into devices for diverse applications.
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.
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 |