The authors acknowledge financial support from the National Research Council of Canada.

<ol>
	<li>Teitell, M., Richardson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+the+immune+system.">DNA methylation in the immune system.</a> Clinical Immunology. 109 (1), 2-5 (2003).</li><li>Suarez-Alvarez, B., Rodriguez, R. M., Fraga, M. F., L&#243;pez-Larrea, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+A+promising+landscape+for+immune+system-related+diseases.">DNA methylation: A promising landscape for immune system-related diseases.</a> Trends in Genetics. 28 (10), 506-514 (2012).</li><li>Su&#225;rez-&#193;lvarez, B., Raneros, A. B., Ortega, F., L&#243;pez-Larrea, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+modulation+of+the+immune+function:+A+potential+target+for+tolerance.">Epigenetic modulation of the immune function: A potential target for tolerance.</a> Epigenetics. 8 (7), 694-702 (2013).</li><li>Kondilis-Mangum, H. D., Wade, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+and+the+adaptive+immune+response.">Epigenetics and the adaptive immune response.</a> Molecular Aspects of Medicine. 34 (4), 813-825 (2013).</li><li>Zouali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Autoimmune Diseases. , (2014).</li><li>Wiencke, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+DNA+methylation+specific+droplet+digital+PCR+(mdPCR)+and+real+time+qPCR+with+flow+cytometry+in+characterizing+human+T+cells+in+peripheral+blood.">A comparison of DNA methylation specific droplet digital PCR (mdPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.</a> Epigenetics. 9 (10), 1360-1365 (2014).</li><li>Hindson, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Analytical Chemistry. 83 (22), 8604-8610 (2011).</li><li>Pinheiro, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+droplet+digital+polymerase+chain+reaction+format+for+DNA+copy+number+quantification.">Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.</a> Analytical Chemistry. 84 (2), 1003-1011 (2012).</li><li>Roy, E., Galas, J. C., Veres, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoplastic+elastomers+for+microfluidics:+Towards+a+high-throughput+fabrication+method+of+multilayered+microfluidic+devices.">Thermoplastic elastomers for microfluidics: Towards a high-throughput fabrication method of multilayered microfluidic devices.</a> Lab on a Chip. 11 (18), 3193-3196 (2011).</li><li>Roy, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cellular+lysis+to+microarray+detection,+an+integrated+thermoplastic+elastomer+(TPE)+point+of+care+Lab+on+a+Disc.">From cellular lysis to microarray detection, an integrated thermoplastic elastomer (TPE) point of care Lab on a Disc.</a> Lab on a Chip. 15 (2), 406-416 (2015).</li><li>Malic, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+subtyping+of+white+blood+cells+using+a+thermoplastic+elastomer-based+microfluidic+emulsification+device+for+multiplexed,+methylation-specific+digital+droplet+PCR.">Epigenetic subtyping of white blood cells using a thermoplastic elastomer-based microfluidic emulsification device for multiplexed, methylation-specific digital droplet PCR.</a> Analyst. 44 (22), 6541-6553 (2019).</li><li>Malic, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-based+microfluidic+chip+for+rapid+and+efficient+immunomagnetic+capture+and+release+of+Listeria+monocytogenes.">Polymer-based microfluidic chip for rapid and efficient immunomagnetic capture and release of Listeria monocytogenes.</a> Lab Chip. 15 (20), 3994-4007 (2015).</li><li>Malic, L., Morton, K., Clime, L., Veres, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-thermoplastic+nanoplasmonic+microfluidic+device+for+transmission+SPR+biosensing.">All-thermoplastic nanoplasmonic microfluidic device for transmission SPR biosensing.</a> Lab Chip. 13 (5), 798-810 (2013).</li></ol>

There are no conflicts to declare.

The presented experimental protocol and methods allow for in-house mdPCR using a fabricated TPE droplet generator, a thermal cycler, and fluorescence microscope. The fabricated device using soft TPE to TPE bonding affords hydrophobic surface properties that are uniform across all channel walls, such that the final device does not require any surface treatment for subsequent use as a droplet generator. This material has been routinely employed in point-of-care platforms that necessitate compatibility with high throughput ...

Analysis of white blood cells (WBCs) composition is among the most frequently requested laboratory tests in hematological diagnostics. Differential leukocyte count serves as an indicator for a spectrum of diseases including infection, inflammation, anemia, and leukemia, and is under investigation as an early prognostic biomarker for several other conditions as well. Gold standard in WBC subtyping involves immunostaining and/or flow cytometry both of which require costly, instability-prone fluorescent antibodies and are often highly dependent on operator proficiency in sample preparation. Moreover, this method is applicable to fresh blood samples only, such that the samples cannot be frozen for shipment or later analysis.
Epigenetic markers have recently emerged as powerful analytical tools for the study of phenotypic variations. Subsequently, human leukocyte populations have been shown to have cell-lineage DNA methylation patterns that allow for the precise characterization of WBC subsets. Subtyping based on epigenetic markers provides a promising alternative that does not depend on fresh blood sample collection or expensive antibodies and can be exploited as a biomarker for disease onset and susceptibility1,2,3,4,5.
Genome-wide studies have been performed for extensive mapping of methylated specific CG-rich regions in the genome (CpG islands) in leukocyte subtypes to identify candidate epigenetic markers specific to leukocyte subtypes. PCR protocols have been developed because of this reason for methylated gene regions, e.g., CD3Z and FOXP3, corresponding to CD3+ T-Cells and CD4+ CD25+ Regulatory T-Cells (T-Regs), respectively. Wiencke et al. have demonstrated the utility of duplex droplet PCR for epigenetic subtyping of T-Cells, yielding results that highly correlate with flow activated cell sorting (FACS) analysis6. This quantitative genetic analysis method relies on partitioning the template nucleic acid molecules and PCR reagents into thousands of discrete, volumetrically defined, sub-nanoliter sized droplets containing zero, one or more target nucleic acid copies, using water-in-oil emulsions enabled by microfluidics7,8. The PCR amplification is performed within each individual droplet and the endpoint fluorescence intensity of each droplet is measured, allowing absolute quantification of targets present in the sample. Droplet PCR has been established to be more precise, accurate, and technically simpler than standard qPCR, making it a more favorable DNA methylation-based method for clinical evaluation of T-Cells. Although a rapidly emerging subtyping methodology, multiplexed epigenetic analysis to probe for various methylated CpG regions simultaneously is lacking. This is necessary for routine leukocyte differential counts.
Herein, a thermoplastic elastomer (TPE) droplet microfluidic device is presented and employed for methylation-specific multiplex droplet PCR (mdPCR). The technology has been used to delineate specific leukocyte subtypes, CD3+ T-Cells and CD4+ CD25+ T-Regs, based on cell-lineage DNA methylation patterns, i.e., epigenetic variation of CD3Z and FOXP3 CpG regions, respectively. A detailed protocol for DNA extraction, bisulfite conversion and mdPCR is described in concert with a fabrication method for a TPE droplet generation device. Representative results of the method are compared to those of immunofluorescence staining highlighting the utility of the proposed approach.

<table>
	<tbody>
		<tr>
			<td>Bio-Rad, Mississauga, ON</td>
			<td>TFI0201</td>
			<td>PCR tube</td>
			<td></td>
		</tr>
		<tr>
			<td>RAN Biotechnologies, Beverly, MA</td>
			<td>008-FluoroSurfactant</td>
			<td>Fluoro-surfactant</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Quest International, Santa Clara, CA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxford Instruments, Abingdon, UK</td>
			<td></td>
			<td>EMCCD camera</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>MA5-16728</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>22-8425-71</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellProfiler</td>
			<td></td>
			<td>Used for fluorescence image analysis</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon, Japan</td>
			<td></td>
			<td>10x objective</td>
			<td></td>
		</tr>
		<tr>
			<td>American Type Culture Collection (ATCC), Manassas, VA</td>
			<td>PCS-800-011</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ram&#233;-Hart Instrument Co. (Netcong, NJ)</td>
			<td>p/n 200-U1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher, Canada</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrocom, NJ, USA</td>
			<td>5015 and 5010</td>
			<td>Borosilicate capilary tube</td>
			<td></td>
		</tr>
		<tr>
			<td>(http://definetherain.org.uk/)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu, Japan</td>
			<td>LC-L1V5</td>
			<td>DEL UV light source</td>
			<td></td>
		</tr>
		<tr>
			<td>Dolomite</td>
			<td>3200063</td>
			<td>Disposable fluidic tubing</td>
			<td></td>
		</tr>
		<tr>
			<td>Dolomite</td>
			<td>3200302</td>
			<td>Disposable fluidic tubing</td>
			<td></td>
		</tr>
		<tr>
			<td>IDT, Coralville, IA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon, Melville, NY</td>
			<td></td>
			<td>Upright light microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytec Industries, Woodland Park, NJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EV Group, Sch&#228;rding, Austria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zymo Research, Irvine, CA</td>
			<td>D5030</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photron, San Diego, CA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IDT, Coralville, IA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gersteltec, Pully, Switzerland</td>
			<td></td>
			<td>SU-8 photoresist</td>
			<td></td>
		</tr>
		<tr>
			<td>Fineline Imaging, Colorado Springs, CO</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen, Hilden, Germany</td>
			<td>203603</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td></td>
			<td>Used to assess droplet diameter</td>
			<td></td>
		</tr>
		<tr>
			<td>Anachemia, Montreal, QC</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excelitas, MA, USA</td>
			<td></td>
			<td>Broad-spectrum LED fluorescent lamp</td>
			<td></td>
		</tr>
		<tr>
			<td>Galenvs Sciences Inc., Montreal, QC</td>
			<td>DE1010</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hexpol TPE, &#197;m&#229;l, Sweden</td>
			<td></td>
			<td>Thermoplastic elastomer (TPE)</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>13-400-518</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon, Japan</td>
			<td></td>
			<td>Used for image acquisition</td>
			<td></td>
		</tr>
		<tr>
			<td>3M, St Paul, MN</td>
			<td></td>
			<td>Carrier Oil</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>R37605</td>
			<td>Blue fluorescent live cell stain (DAPI)</td>
			<td></td>
		</tr>
		<tr>
			<td>IDEX Health &#38; Science, Oak Harbor, WA</td>
			<td>P-881</td>
			<td>PEEK fittings</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma-Aldrich, Oakville, ON</td>
			<td>806552</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning, Midland, MI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ThinkyUSA, CA, USA</td>
			<td>ARV 310</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ihc world, Maryland, USA</td>
			<td>IW-125-0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zinsser NA, Northridge, CA</td>
			<td>2607808</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cetoni GmbH, Korbussen, Germany</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma-Aldrich, Oakville, ON</td>
			<td>484431</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad, Mississauga, ON</td>
			<td>1861096</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hitachi High-Technologies, Mississauga, ON</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon, Melville, NY</td>
			<td></td>
			<td>Inverted microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon, Japan</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Loctite</td>
			<td>AA 352</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

methylation specific multiplex droplet pcr using polymer droplet generator device for hematological diagnostics

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC&#160;subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

The TPE-based microfluidic droplet generator device was fabricated using the described protocol as shown in Figure 1. A transparency mask was used in photolithography to obtain silicon (Si) master. Soft lithography was performed to obtain an inverse PDMS replica of the Si master which was then used to fabricate the epoxy mold. Epoxy precursor was poured onto the PDMS and cured to crosslink and harden. This mold, representing the exact replica of the Si master was more resilient for subsequen...

Watch this Scientific Journal Video about Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics at JoVE.com

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, ...

methylation-specific-multiplex-droplet-pcr-using-polymer-droplet

Research

JoVE Journal

Bioengineering

5.1K Views.  National Research Council of Canada. Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.

Video: Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Bulk Droplet Vitrification for Primary Hepatocyte Preservation

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. Vitrification requires extremely fast cooling rates to achieve transition of water into the glass phase while avoiding injurious ice formation. Although pre-incubation with cryoprotective agents (CPA) can reduce the critical cooling rate of biological samples, high concentrations are generally needed to enable ice-free cryopreservation by vitrification. As a result, vitrification is hampered by CPA toxicity and restricted to small samples that can be cooled fast. It was recently demonstrated that these inherent limitations can be overcome by bulk droplet vitrification. Using this novel method, cells are first pre-incubated with a low intracellular CPA concentration. Leveraging rapid osmotic dehydration, the intracellular CPA is concentrated directly ahead of vitrification, without the need to fully equilibrate toxic CPA concentrations. The cellular dehydration is performed in a fluidic device and integrated with continuous high throughput generation of large sized droplets that are vitrified in liquid nitrogen. This ice-free cryopreservation method with minimal CPA toxicity is suitable for large cell quantities and results in increased hepatocyte viability and metabolic function as compared to classical slow-freezing cryopreservation. This manuscript describes the methods for successful bulk droplet vitrification in detail.

This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...