This work was supported by National Research Foundation of Korea (NRF) grants (2013R1A2A2A05004314, 2012R1A1A2043747), a grant from the Korean Health Technology R&#38;D Project, Ministry of Health &#38; Welfare (A121994) and IBS-R020-D1 funded by the Korean Government.

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
<ol>
	<li>Preparation of precursor solution22
	<ol>
		<li>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 &#176;C) for 30 min on a magnetic stirrer.</li>
		<li>Dissolve 11 wt% of polyvinyl pyrrolidone (PVP) in 3.64 g of ethanol (99.9%) and stir the solution at 65 &#176;C for 1 hr using a hot-plate magnetic stirrer.</li>
		<li>Combine and stir the two solutions at 65 &#176;C for 4 hr on a hot-plate magnetic stirrer.</li>
	</ol>
	</li>
	<li>Electrospinning
	<ol>
		<li>Load the prepared solution into a syringe connected to a stainless steel needle of 200 &#956;m inner diameter. Pour the solution directly into the syringe after removing the plunger.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Calcination of the NF mat
	<ol>
		<li>Place the NF mat in a furnace and increase the temperature up to 500 &#176;C with a ramping rate of 3 &#176;C/min under high-vacuum condition (5 x 10-5 torr).</li>
		<li>Maintain the temperature at 500 &#176;C for 3 hr. Cool down the temperature of the samples to the RT (25 &#176;C) in the furnace.</li>
	</ol>
	</li>
</ol>
2. Integration of TiO2 NF Mat into a Centrifugal Microfluidic Disc
<ol>
	<li>Fabrication of a disc
	<ol>
		<li>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).</li>
		<li>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: &#34;AUTO&#34; -&#62; &#34;G CODE OPEN&#34; -&#62; Select the generated CNC code.</li>
		<li>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 &#34;START&#34; button.</li>
	</ol>
	</li>
	<li>Transfer of TiO2 NF mat onto the disc
	<ol>
		<li>Place a silicon substrate and a small petri dish containing 40 &#181;L 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.</li>
		<li>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.</li>
		<li>Cure the PDMS coated on silicon substrate in an oven at 65 &#176;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.</li>
		<li>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 &#176;C for 4 hr or at 80 &#176;C for 1 hr to cure the PDMS layer completely.</li>
		<li>Coat binding reaction chambers in the disc with 20 &#181;l&#160;the PDMS/curing agent mixture (10:1), which acts as an adhesive layer to attach TiO2 NF mat onto the disc.</li>
		<li>Dispense 50 &#181;l 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 &#181;l 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.</li>
		<li>Incubate the TiO2 NF mat integrated disc in the oven at 65 &#176;C for 4 hr or at 80 &#176;C for 1 hr to cure the PDMS completely.</li>
	</ol>
	</li>
	<li>Surface modification of the TiO2 NF mat on the disc 
	<ol>
		<li>Prepare 1% v/v solution of (3-glycidoxypropyl)methyldiethoxy silane (GPDES) in ethanol (99.9%).</li>
		<li>Treat the TiO2 NF mat integrated disc with oxygen plasma at 140 W, 50 sccm oxygen flow for 180 sec. Dispense 100 &#956;l of GPDES solution on each nanofiber mat and incubate at RT (25 &#176;C) for 2 hr.</li>
		<li>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 &#176;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.</li>
		<li>Blow dry with a nitrogen stream, dry under vacuum. 
		NOTE: The disc can be stored in a sealed container at RT (25 &#176;C) until use.</li>
	</ol>
	</li>
	<li>Immobilization of antibodies on the surface
	<ol>
		<li>Make a solution of 200 &#956;g/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 &#956;l of the solution onto each NF mat in a disc using a micropipette. See materials list for more information about the antibodies.</li>
		<li>Keep the discs in a humidified chamber and incubate at 37 &#176;C for 4 hr. Wash the antibodies&#160;coated NF mat with 0.1% BSA-PBS buffer. Fill the chamber with 100 &#956;l 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.</li>
	</ol>
	</li>
	<li>Disc assembly
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Immunoassay
<ol>
	<li>Fill the chambers with 1% BSA-PBS buffer using a micropipette and incubate the disc at 37 &#176;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.</li>
	<li>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 &#176;C until use.</li>
	<li>Load 10 &#956;l 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 &#181;M range, CRP-free serum was used to demonstrate the low detection limit of the device.</li>
	<li>Spin the disc at 3,600 rpm (391 x g) for 60 sec to separate the red blood cells.</li>
	<li>Open valve #1 by laser irradiation, and transfer 4 &#956;l of the supernatant plasma to the chamber containing 8 &#956;l 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</li>
	<li>Apply a mixing mode (15 Hz s-1, 15&#176;) for 5 sec for binding of the protein and detection antibodies.</li>
	<li>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&#176;) 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).</li>
	<li>Transfer the washing buffer (600 &#181;l) 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&#176;) for 120 sec to wash the TiO2 NFs in the chamber.</li>
	<li>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.</li>
	<li>For the chemiluminescent reaction, apply a mixing mode (30 Hz s-1, 2&#176;) 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.</li>
	<li>Transfer the reacted substrate solution to the detection chambers&#160;by opening valve #7 and spinning the disc at 2,400 rpm for 10 sec.</li>
	<li>Measure chemiluminescence signals from the reacted substrate solution using a photomultiplier tube (PMT) module equipped detection system. &#160; 
	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.</li>
</ol>

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The authors have nothing to disclose.

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 &#956;l) 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...

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 &#956;L 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 &#956;l&#160;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.

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

fully automated centrifugal microfluidic device for ultrasensitive protein detection from whole blood

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.

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

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L 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 ...

fully-automated-centrifugal-microfluidic-device-for-ultrasensitive

Research

JoVE Journal

Bioengineering

10.4K Views.  School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan, Republic of Korea. 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.

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

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Engineering

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection

Source: Sage, J. M., et al. <a href="https://app.jove.com/v/60638">Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a capillary electrophoresis immunoassay protocol for detecting a protein biomarker associated with amyotrophic lateral sclerosis (ALS) from human blood platelets. Confirmation of the presence of the ALS biomarker protein is achieved through size-based separation of the platelet proteins followed by chemiluminescent detection.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of buffers and ...

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

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