Method Article

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

DOI:

10.3791/54143

⸱

April 16th, 2016

 ,  ,  ,  ,  , 
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)

In This Article

Summary

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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.

Abstract

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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

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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.

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Protocol

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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. 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 magnetic stirrer.
    3. Combine and stir the two solutions at 65 °C for 4 hr on a hot-plate magnetic stirrer.
  2. Electrospinning
    1. Load the prepared solution into a syringe connected to a stainless steel needle of 200 μm inner diameter. Pour the solution directly into the syringe after removing the plunger.
    2. Introduce the solution constantly at a flow rate of 0.3 ml/hr for 10 min onto a Si wafer (2 cm x 2 cm) placed on a grounded substrate at a high DC voltage (15 kV). During the electrospinning, set the distance between the needle and the grounded substrate to 10 cm.
  3. Calcination of the NF mat
    1. Place the NF mat in a furnace and increase the temperature up to 500 °C with a ramping rate of 3 °C/min under high-vacuum condition (5 x 10-5 torr).
    2. Maintain the temperature at 500 °C for 3 hr. Cool down the temperature of the samples to the RT (25 °C) in the furnace.

2. Integration of TiO2 NF Mat into a Centrifugal Microfluidic Disc

  1. Fabrication of a disc
    1. Use the 3D designing software (e.g., SolidWorks or similar) to depict chambers, channels, or valves of each layer of disc. Convert the design files to computer numerical control (CNC) code using code generating software (e.g., Edgecam or similar). Use the softwares according to the instruction manual.23,24 Note: The typical channel dimensions used in this device are 1.0 mm x 0.3 mm (width x depth).
    2. Open the operating software of CNC milling machine (e.g., DeskCNC or similar) and load CNC code to the operating software and click the following in order: "AUTO" -> "G CODE OPEN" -> Select the generated CNC code.
    3. Attach the PC plate on the CNC milling machine: use 1 mm PC plates for the top layer and 5 mm PC plates for the disc layer. Run the CNC milling machine to cut each layer of the disc by clicking the "START" button.
  2. Transfer of TiO2 NF mat onto the disc
    1. Place a silicon substrate and a small petri dish containing 40 µL of (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane in a vacuum chamber under vacuum for 30 min. The fluorosilane vaporizes and gets coated on the substrate.
    2. Mix polydimethylsiloxane (PDMS) pre-polymer with a curing agent in a 10:1 ratio and degas in a vacuum chamber. Spin-coat the PDMS onto a silicon substrate at 650 rpm for 60 sec.
    3. Cure the PDMS coated on silicon substrate in an oven at 65 °C for 10 min to achieve an adherent state.
      NOTE: Curing time can be varied depending on environmental conditions, and adhesion force can be tested by a tack test using a rheometer.
    4. Transfer the TiO2 NF mat prepared by electrospinning and calcination onto the PDMS-coated silicon using a tweezer. Then, manually apply a conformal pressure on the transferred TiO2 NF mat with a flat object (e.g., glass slide) and remove the object. Put this sample in the oven at 65 °C for 4 hr or at 80 °C for 1 hr to cure the PDMS layer completely.
    5. Coat binding reaction chambers in the disc with 20 µl the PDMS/curing agent mixture (10:1), which acts as an adhesive layer to attach TiO2 NF mat onto the disc.
    6. Dispense 50 µl of ethanol (99.9%) on the TiO2 NF mat on the PDMS layer, cut the NF mat with a punch hole of 6 mm diameter, and transfer the cut TiO2 NF mat to the binding reaction chambers coated with 20 µl of the PDMS in the previous step.
      NOTE: In this step, ethanol will be helpful to detach the cut TiO2 NF mat from the Si substrate.
    7. Incubate the TiO2 NF mat integrated disc in the oven at 65 °C for 4 hr or at 80 °C for 1 hr to cure the PDMS completely.
  3. Surface modification of the TiO2 NF mat on the disc
    1. Prepare 1% v/v solution of (3-glycidoxypropyl)methyldiethoxy silane (GPDES) in ethanol (99.9%).
    2. Treat the TiO2 NF mat integrated disc with oxygen plasma at 140 W, 50 sccm oxygen flow for 180 sec. Dispense 100 μl of GPDES solution on each nanofiber mat and incubate at RT (25 °C) for 2 hr.
    3. Wash the substrates briefly by dispensing ethanol (99.9%) using a wash bottle, then remove the ethanol completely by inverting the disc and blotting it against a clean wipe. Cure at 80 °C for 1 hr. Wash twice with ethanol (99.9%) in the same manner as stated above, to remove the physically adsorbed and unbound GPDES molecules.
    4. Blow dry with a nitrogen stream, dry under vacuum.
      NOTE: The disc can be stored in a sealed container at RT (25 °C) until use.
  4. Immobilization of antibodies on the surface
    1. Make a solution of 200 μg/ml of capture antibodies (monoclonal mouse antihuman hsCRP or monoclonal mouse anti-cTnI) by diluting the antibodies with a phosphate buffered saline (PBS) buffer (pH 7.4). Dispense 5 μl of the solution onto each NF mat in a disc using a micropipette. See materials list for more information about the antibodies.
    2. Keep the discs in a humidified chamber and incubate at 37 °C for 4 hr. Wash the antibodies coated NF mat with 0.1% BSA-PBS buffer. Fill the chamber with 100 μl of wash buffer using a micropipette, remove the buffer by aspirating or decanting. Finally, invert the disc and blot it against a clean wipe, and then assemble the disc.
  5. Disc assembly
    1. Draw the design of the disc on double-side adhesive tape using a CAD program (e.g., AutoCAD or similar). Load the CAD design to the cutting plotter.
    2. Cut the double-side adhesive tape using the cutting plotter. Peel off one protection layer of double adhesive tape and attach it on top of the disc layer. Peel off the other protection layer and attach the top layer on the disc layer.
    3. Load the preliminarily assembled disc in the pressing machine and precisely align top/adhesive/disc layers using align marks in each layer to connect each valve, channel, and chambers. Apply conformal pressure using the pressing machine.

3. Immunoassay

  1. Fill the chambers with 1% BSA-PBS buffer using a micropipette and incubate the disc at 37 °C for 1 hr. Remove the buffer by aspiration using a micropipette. Perform this step to block the un-reacted sites and to reduce non-specific adsorption of protein in a disc.
  2. Wash twice with 0.1% BSA-PBS by filling and aspirating the chambers using a micropipette.
    NOTE: At this stage the disc can be stored at 4 °C until use.
  3. Load 10 μl of antigen-spiked whole blood or CRP-free serum for CRP detection on the disc using a micropipette. For making the calibration graphs, use concentrations of CRP from 1 pg/ml to 100 ng/ml; and cTnI from 10 pg/ml to 100 ng/ml.
    NOTE: Due to the higher levels of CRP in whole blood, which is usually in µM range, CRP-free serum was used to demonstrate the low detection limit of the device.
  4. Spin the disc at 3,600 rpm (391 x g) for 60 sec to separate the red blood cells.
  5. Open valve #1 by laser irradiation, and transfer 4 μl of the supernatant plasma to the chamber containing 8 μl of detecting antibodies conjugated with HRP by spinning the disc at 2,400 rpm for 3 sec.
    NOTE: The general processes for valve actuation and visualization of disc operation are described in detail in a previous report.21
  6. Apply a mixing mode (15 Hz s-1, 15°) for 5 sec for binding of the protein and detection antibodies.
  7. Open valve #2, and transfer the mixture prepared in step 6 to the binding reaction chamber (2,400 rpm, 3 sec). Then, apply a mixing mode (60 Hz s-1, 2°) for 20 min to achieve an immunoreaction between the mixture and the binding antibodies on the TiO2 NFs. After the reaction, open valve #3 and transfer the residual mixture to the waste chamber (2,400 rpm, 10 sec).
  8. Transfer the washing buffer (600 µl) to the binding reaction chamber by opening valve #4 and spinning the disc (2,400 rpm, 4 sec). Then apply a mixing mode (30 Hz s-1, 30°) for 120 sec to wash the TiO2 NFs in the chamber.
  9. Wash the TiO2 NFs twice more by following same condition of step 3.8, and remove the residual washing buffer completely by spinning the disc at 2,400 rpm for 20 sec. Then, close the valve #5.
  10. For the chemiluminescent reaction, apply a mixing mode (30 Hz s-1, 2°) for 1 min after transferring the chemiluminescent substrate solution to the binding reaction chamber by opening valve #6 and spinning the disc at 2,400 rpm for 10 sec subsequently.
  11. Transfer the reacted substrate solution to the detection chambers by opening valve #7 and spinning the disc at 2,400 rpm for 10 sec.
  12. Measure chemiluminescence signals from the reacted substrate solution using a photomultiplier tube (PMT) module equipped detection system.  
    NOTE: The detection system is a custom-built machine using the following parts: step motor, optomechanical components, translation stages, and commercially available PMT. The optomechanical parts and step motor are assembled to hold the device, and the motor can rotate the disc. The detection is performed by PMT fixed on the optomechanical parts, and it is located above the detection chambers of the device. By manipulating the step motor, the detection chambers are aligned right below the detection zone of PMT.

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Results

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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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Discussion

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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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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Si waferLG SILTRONPolished Wafer, test gradeDia. (mm) = 150, orientation = <100>, dopant = boron, RES(Ohm-cm) = 1 - 30, thickness (μm) = 650 - 700
Polycarbonate (PC) Daedong PlasticPCS#6900Thickness (mm) = 1 and 5 
Titanium tetraisopropoxide, 98%,Sigma-Aldrich205273
Polyvinylpyrrolidone, Mw = 1,300,000Sigma-Aldrich437190
Acetic acidSigma-Aldrich320099
Anhydrous ethanolSigma-Aldrich459836
Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilaneSigma-Aldrich448931
PDMS and curing agentDow CorningSYLGARD 184
GPDESGelest IncSIG5832.0 
EthanolJ T Baker
FE-SEMFEINova NanoSEM
X-ray photoelectron spectroscopyThermoFisherK-alpha
3D modeling machineM&I CNC Lab, KoreaCNC milling machine
Wax-dispensing machineHanra Precision Eng. Co. Ltd., KoreaCustomized
Double-sided adhesive tapeFLEXcon, USADFM 200 clear 150 POLY H-9 V-95
Cutting plotterGraphtec Corporation, JapanGraphtec CE3000-60 MK2
Spin coaterMIDASSPIN-3000D
Furnace (calcination)R. D. WEBB COMPANYWEBB 99
Rheometer (Tack test)Thermo ScientificHaake MARS III - ORM Package
Oxygen plasma systemFEMTOCUTE
Monoclonal mouse antihuman hsCRPHytest Ltd., Finland4C28(clone # C5)
Monoclonal mouse anti-cTnIHytest Ltd., Finland4T21(clone # 19C7)
HRP conjugated goat polyclonal anti-hsCRPAbcam plc., MAab19175
HRP conjugated mouse monoclonal anti-cTnIAbcam plc., MAab24460(clone # 16A11)
hsCRPAbcam plc., MAab111647
cTnIFitzgerald, MA30-AT43
Bovine AlbuminSigma-AldrichA7906
PBSAmresco IncE404
Blood collection tubesBD vacutainer367844K2 EDTA 7.2 mg plus blood
collection tubes
SuperSignal ELISA femtoInvitrogen37074
Modular multilabel plate readerPerkin ElmerEnvision 2104
Disc operating machineHanra Precision Eng. Co. Ltd., KoreaCustomized
Photomultiplier tube (PMT)Hamamatsu PhotonicsH1189-210
AutoCADAutoDeskVersion 2012Design software
SolidWorks 3D CAD software SOLIDWORKS Corp.Version 20133D Design software,
EdgecamVero softwareversion 2009.01.06928Code generating software
DeskCNCCarken Co.version 2.0.2.18CNC milling machine software

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ELISANanofiber IntegrationElectrospinningPDMS CoatingDisc AssemblyAutomated ImmunoassayFemtomolar Sensitivity

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