This research was supported by National Institutes of Health (NIH) grant R01 EB008268.

1. Chip Fabrication12
<ol>
 <li>Make two plaques from Zeonex 690R pellets: distribute 8-9 grams Zeonex pellets evenly in the center of a metal plate, preheat on the heated press at 198 &deg;C for 5 min, and then apply pressure slowly to 2,500 psi for another 5 min. To complete this step, we used a Carver hot press.</li>
 <li>Emboss the microfluidic channel in the plaque with an epoxy mold. Details on the mold fabrication are outlined elsewhere12 (channel design in Figure 2b</str...

<ol>
	<li>Nicholls, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandemic+Influenza:+The+Inside+Story.">Pandemic Influenza: The Inside Story.</a> PLoS Biol. 4, (2006).</li><li>Wenzel, J. 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=Analytical+performance+determination+and+clinical+validation+of+the+novel+roche+realtime+ready+influenza+A/H1N1+detection+set.">Analytical performance determination and clinical validation of the novel roche realtime ready influenza A/H1N1 detection set.</a> Journal of Clinical Microbiology. 48, 3088-3094 (2010).</li><li>Chan, K. H., 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=Analytical+sensitivity+of+rapid+influenza+antigen+detection+tests+for+swine-origin+influenza+virus+(H1N1.">Analytical sensitivity of rapid influenza antigen detection tests for swine-origin influenza virus (H1N1.</a> Journal of Clinical Virology: the. 45, 205-207 (2009).</li><li>Ginocchio, C. C., 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+multiple+test+methods+for+the+detection+of+the+novel+2009+influenza+A+(H1N1)+during+the+New+York+City+outbreak.">Evaluation of multiple test methods for the detection of the novel 2009 influenza A (H1N1) during the New York City outbreak.</a> Journal of. 45, 191-195 (2009).</li><li>Aeron, C. H., 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=Performance+of+influenza+rapid+point-of-care+tests+in+the+detection+of+swine+lineage+A(H1N1)+influenza+viruses.">Performance of influenza rapid point-of-care tests in the detection of swine lineage A(H1N1) influenza viruses.</a> Influenza and Other Respiratory Viruses. 3, 171-176 (2009).</li><li>Takahashi, H., Otsuka, Y., Patterson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+for+influenza+and+other+respiratory+viruses:+determining+performance+specifications+based+on+clinical+setting.">Diagnostic tests for influenza and other respiratory viruses: determining performance specifications based on clinical setting.</a> Journal of Infection and Chemotherapy. 16, 155-161 (2010).</li><li>Selvaraju, S. B., Selvarangan, R. <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+Three+Influenza+A+and+B+Real-Time+Reverse+Transcription-PCR+Assays+and+a+New+2009+H1N1+Assay+for+Detection+of+Influenza+Viruses.">Evaluation of Three Influenza A and B Real-Time Reverse Transcription-PCR Assays and a New 2009 H1N1 Assay for Detection of Influenza Viruses.</a> Journal of Clinical Microbiology. 48, 3870-3875 (2009).</li><li>Northrup, M. A., Ching, M. T., White, R. M., Watson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transducer'93, seventh international conference on solid state sensors and actuators. , 924-926 (1993).</li><li>Bhattacharyya, A., Klapperich, C. 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C., Klapperich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+microfluidic+chip+for+continuous-flow+polymerase+chain+reaction:+Simulations+and+experiments.">Plastic microfluidic chip for continuous-flow polymerase chain reaction: Simulations and experiments.</a> Biotechnology Journal. 6, 177-184 (1002).</li><li>Cao, Q., 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=Microfluidic+Chip+for+Molecular+Amplification+of+Influenza+A+RNA+in+Human+Respiratory+Specimens.">Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens.</a> PLoS ONE. 7, e33176 (2012).</li><li>R&#229;dstr&#246;m, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+Characterization+of+PCR-Inhibitory+Components+in+Blood+Cells.">Purification and Characterization of PCR-Inhibitory Components in Blood Cells.</a> J. Clin. Microbiol. 39, 485-493 (2001).</li><li>Boddinghaus, B., Wichelhaus, T. A., Brade, V., Bittner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+PCR+Inhibitors+by+Silica+Membranes:+Evaluating+the+Amplicor+Mycobacterium+tuberculosis+Kit.">Removal of PCR Inhibitors by Silica Membranes: Evaluating the Amplicor Mycobacterium tuberculosis Kit.</a> J. Clin. Microbiol. 39, 3750-3752 (2001).</li><li>Andreasen, D., 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=Improved+microRNA+quantification+in+total+RNA+from+clinical+samples.">Improved microRNA quantification in total RNA from clinical samples.</a> Methods. 50, S6-S9 (2010).</li></ol>

The diagnostic method presented here demonstrated the ability of an integrated microfluidic plastic chip to amplify influenza A RNA from patient specimens with high specificity and a low detection limit.13 We designed this chip for potential point of care testing: (a) the temperature and fluidic control were simplified, (b) the chip is low cost and suitable for high throughput fabrication using injection molding, and (c) the chip is disposable and intended for one time use, thus reducing the concern of s...

Millions of deaths have been reported during the three influenza pandemics of the 20th century1. Moreover, the most recent influenza pandemic was declared by World Health Organization (WHO) 2 in 2009, and as of August 1, 2010, 18,449 deaths were reported by WHO3. This pandemic demonstrated again the high burden of infectious disease, and the need for rapid and accurate detection of influenza to enable fast disease confirmation, appropriate public health response and effective treatment4.
There are several methods widely used for diagnosing influenza, these include rapid immunoassays, direct fluorescent antigen testing (DFA) and viral culture methods. Rapid immunoassays dramatically lack sensitivity5-8, while the other two methods are labor-intensive and time consuming9. Molecular tests offer multiple advantages including a short turn-around time, high sensitivity, and higher specificity. Several commercial entities have been working towards fast molecular tests (also called nucleic acid tests or NATs) for infectious diseases, and several have influenza assays in their pipelines. However most of them require off-chip sample preparation. None of the Clinical Laboratory Improvement Amendments (CLIA) waived molecular tests incorporate sample preparation into the assay cartridge or module.
Lab-on-a-chip technology plays an important role in the development of point-of-care testing devices. After the introduction of the first PCR chip in 199310, numerous efforts have been put into developing nucleic acid test chips. However, only a few of these have integrated crude sample preparation with downstream amplification.
We have previously demonstrated the miniaturization of a solid phase extraction column (SPE) into a plastic microfluidic chip11 and the development and optimization of a continuous flow PCR chip12. Here, we extend the previous work to integrate the SPE with RT and PCR steps into a single chip for clinical diagnostics and demonstrate its capability to amplify nucleic acids from patient nasopharyngeal (NP) swabs and aspirates.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>1-dodecanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>443816-500G</td>
 </tr>
 <tr>
 <td>2,2-Dimethoxy-2-phenylacetophenone</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>196118-50G</td>
 </tr>
 <tr>
 <td>2100 Bioanalyzer</td>
 <td>Agilent Technologies, Santa Clara, CA</td>
 <td>G2943CA</td>
 </tr>
 <tr>
 <td>2-Propanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>19516</td>
 </tr>
 <tr>
 <td>Benzophenone</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>239852-50G</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Thermo Fisher Scientific,pittsburge, PA</td>
 <td>A7979-50ML</td>
 </tr>
 <tr>
 <td>Butyl methacrylate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>235865-100 ml</td>
 </tr>
 <tr>
 <td>Carrier RNA</td>
 <td>Qiagen, Valencia, CA</td>
 <td>1017647</td>
 </tr>
 <tr>
 <td>Cyclohexanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>105899-1L</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>E7023</td>
 </tr>
 <tr>
 <td>Ethylene dimethacrylate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>335861</td>
 </tr>
 <tr>
 <td>Ethylene glycol dimethacrylate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>335681-100ML</td>
 </tr>
 <tr>
 <td>Glass syringe 250 &mu;l</td>
 <td>Hamilton, Reno, NV </td>
 <td>81127</td>
 </tr>
 <tr>
 <td>Guanidine thiocyanate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>50981</td>
 </tr>
 <tr>
 <td>High Sensitivity DNA Kit</td>
 <td>Agilent Technologies, Santa Clara, CA</td>
 <td>5067-4626 </td>
 </tr>
 <tr>
 <td>Hot press</td>
 <td>Carver,Wabash, IN</td>
 <td>4386</td>
 </tr>
 <tr>
 <td>J-B Weld Epoxies</td>
 <td>Mcmaster-Carr,Elmhurst, IL </td>
 <td>7605A11</td>
 </tr>
 <tr>
 <td>Luer-Lok syringes</td>
 <td>BD-Medical, Franklin Lakes, NJ</td>
 <td>309628</td>
 </tr>
 <tr>
 <td>Magnesium Chloride</td>
 <td>Thermo Fisher Scientific,pittsburge, PA</td>
 <td>AB-0359</td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>494437</td>
 </tr>
 <tr>
 <td>Methyl methacrylate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>M55909</td>
 </tr>
 <tr>
 <td>Nanoport</td>
 <td>Upchurch Scientific</td>
 <td>N-333-01</td>
 </tr>
 <tr>
 <td>Nanoport Fitting</td>
 <td>Upchurch Scientific</td>
 <td>F-120x</td>
 </tr>
 <tr>
 <td>Nuclease free water</td>
 <td>Thermo Fisher Scientific,pittsburge, PA</td>
 <td>PR-P1193</td>
 </tr>
 <tr>
 <td>OneStep RT-PCR kit</td>
 <td>Qiagen, Valencia, CA</td>
 <td>210210</td>
 </tr>
 <tr>
 <td>PEG8000</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>41009</td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>VWR,Radnor, PA</td>
 <td>300V</td>
 </tr>
 <tr>
 <td>RNAse Away</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>83931-250ML</td>
 </tr>
 <tr>
 <td>RNASecure</td>
 <td>Applied Biosystems, Foster City, CA</td>
 <td>AM7005</td>
 </tr>
 <tr>
 <td>Silica microspheres</td>
 <td>Polysciences,Warrington, PA</td>
 <td>24324-15</td>
 </tr>
 <tr>
 <td>Syringe pump </td>
 <td>Harvard Apparatus,Holliston, MA</td>
 <td>HA2000P/10</td>
 </tr>
 <tr>
 <td>Thermally Conductive Tape</td>
 <td>Mcmaster-Carr,Elmhurst, IL </td>
 <td>6838A11</td>
 </tr>
 <tr>
 <td>Thermocouple</td>
 <td>Omega Engineering, Stamford, CT</td>
 <td>5SRTC-TT-J-40-36</td>
 </tr>
 <tr>
 <td>Thin-film Heaters</td>
 <td>Minco,Minneapolis, MN </td>
 <td>HK5166R529L12A</td>
 </tr>
 <tr>
 <td>Ultraviolet Crosslinker</td>
 <td>UPV, Upland, CA</td>
 <td>CL-1000</td>
 </tr>
 <tr>
 <td>Zeonex</td>
 <td>Zeon Chemicals, Louisville, KY</td>
 <td>690R</td>
 </tr>
 </tbody>
</table>

microfluidic chip fabrication and method to detect influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

A typical result is shown in Figure 3 for an influenza A infected nasopharyngeal wash specimen. Due to the different amounts of influenza virus in each patient specimen, the final concentration of PCR product will vary. A good result should have low noise, two clear ladder peaks (35 and 10380 bp) and a single product peak at the designed product size (107 bp) for the positive sample. While the product peak should theoretically be absent for negative controls, we did observe spurious PCR peaks near...

Watch this Scientific Journal Video about Microfluidic Chip Fabrication and Method to Detect Influenza at JoVE.com

Microfluidic Chip Fabrication and Method to Detect Influenza

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

microfluidic-chip-fabrication-and-method-to-detect-influenza

Research

JoVE Journal

Bioengineering

14.8K ビュー.  Boston University. 統合されたマイクロ流体チップは、熱可塑性の分子診断として使用するために開発されました。チップは、核酸抽出、逆転写酵素、およびPCRを行います。チップを製造して実行するための方法が記載されている。

ビデオ: Microfluidic Chip Fabrication and Method to Detect Influenza

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are...

a microfluidic chip for icpms sample introduction

A Microfluidic Chip for ICPMS Sample Introduction

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000...

dry film photoresist-based electrochemical microfluidic biosensor platform: device fabrication, on-chip assay preparation, and system operation

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

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...

chip-based digital pcr to detect rare transcript variants using a nanofluidic chip

This video demonstrates chip-based digital PCR &#8212; a variation of the digital PCR technique that is useful in detecting rare transcript variants. The PCR reaction is partitioned into the chambers of a nanofluidic chip, each of which acts as an independent reaction. The detection of fluorescence signals from the chambers with amplified targets confirms the presence of rare transcript variants in the...

Chip-Based Digital PCR to Detect Rare Transcript Variants Using a Nanofluidic Chip

fabrication and testing of microfluidic optomechanical oscillators

Fabrication and Testing of Microfluidic Optomechanical Oscillators

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

Then, for each reaction, add 0.87 microliters of the custom assay primer, and add 1.83 microliters of nuclease-free water. Mix the combination gently, and transfer 11.4 microliters to each tube containing cDNA, and to the no template control. Then, mix the tubes gently and pool the tube contents using pulse centrifugation. For optimal results, load the chips as soon as...

a microfluidic chip-based method for rapid neutrophil chemotaxis analysis using whole blood

This video demonstrates neutrophil chemotaxis assay using an all-on-chip method. The method allows direct isolation of neutrophils from the whole blood by immunomagnetic negative selection followed by chemotaxis assay on a microfluidic...

A Microfluidic Chip-Based Method for Rapid Neutrophil Chemotaxis Analysis Using Whole Blood

Next, remove the Fibronectin solution from the device, and add 100 microliters of migration medium from the outlet. Again, wait three minutes to ensure that all of the channels are filled with the migration medium. Incubate the device for another hour at room temperature, before using the device in the chemotaxis experiment. Place 10 microliters of whole blood into a 1.5-milliliter tube. Then, add 2 microliters of the antibody cocktail in two microliters of magnetic particles from a neutrophil...

a simple microfluidic chip for long-term growth and imaging of caenorhabditis elegans

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

solvent bonding for fabrication of pmma and cop microfluidic devices

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices

Solvent bonding is a simple and versatile method for fabricating thermoplastic microfluidic devices with high quality bonds. We describe a protocol to achieve strong, optically clear bonds in PMMA and COP microfluidic devices that preserve microfeature details, by a judicious combination of pressure, temperature, an appropriate solvent, and device...

rapid fabrication of custom microfluidic devices for research and educational applications

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational...

fabrication of the thermoplastic microfluidic channels

Fabrication of the Thermoplastic Microfluidic Channels

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase...

scalable fabrication of stretchable, dual channel, microfluidic organ chips

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in...

microfluidic fabrication of polymeric and biohybrid fibers with predesigned size and shape

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and...

a technique to detect cyanobacterial strains using an antibody microarray chip

This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct...

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

fabrication and validation of an organ-on-chip system with integrated electrodes to directly quantify transendothelial electrical resistance

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally...

a microfluidic chip for the versatile chemical analysis of single cells

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

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...

rapid detection of bacterial pathogens causing lower respiratory tract infections via microfluidic-chip-based loop-mediated isothermal amplification

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

https://cloudfront.jove.com/CDNSource/teasers/66677_b.jpg

Following slide and cassette assembly, pipette up to 50 microliters of each sample extract or dilution of it in extraction incubation buffer into each well of the cassette. Alternatively, load samples into other incubation devices or simply use cover...

microfluidic picoliter bioreactor for microbial single-cell analysis: fabrication, system setup, and operation

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional...

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 ...

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

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 ...

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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 ...

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...