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): put one plaque onto the epoxy mold; preheat them on the heated press at 157 &deg;C for 10 min and then apply pressure slowly to 1,000 psi for another 10 min. Remove the plaque from the mold by hand or using a tongs before it cools down. (Note: wear thermal gloves during this step.)</li>
 <li>Drill holes in the embossed chip at the inlet, waste port, and the outlet of the microfluidic channel. We used a 1.25 mm diameter drill bit to make the three holes.</li>
 <li>Bond the embossed chip with another plaque (on top): Wash the chips with IPA, RNAse Away and deionized water in sequence; Air dry and preheat them on the heated press at 131 &deg;C for 10 min and then press at 350 psi for another 10 min.</li>
 <li>Attach Nanoports at the inlet, waste, and the outlet ports separately using J-B Weld Epoxy; cure for 15 to 24 hr as per the manufacturer's instructions.</li>
 <li>Rinse the SPE channel with 50 &mu;l RNAse Away, followed with 100 &mu;l nuclease free water.</li>
 <li>Load the SPE channel with 4 &mu;l of the grafting solution (methyl methacrylate with 3% w/v benzophenone) and then crosslink the methacrylate by incubating for 10 min in a UV oven under 365 nm UV wavelength and 2,000 mJ/cm2. Remove the residual grafting solution with vacuum. We used the wall vacuum. For the best results, a fresh solution should be used. It is usually acceptable, however, to store a prepared solution at 4 &deg;C for up to one week.</li>
 <li>Make the SPE column solution (all v/v %): 16% ethylene dimethacrylate, 24% butyl methacrylate, 42% 1-dodecanol, 18% cyclohexanol, and add 1% 2-dimethylamino-4-(methyl-phenylamino)-phenol as the photo-initiator.</li>
 <li>Take out the same volume of silica microspheres solution as the SPE column solution (to make a 1:1 mixture), and completely dry it at 70 &deg;C overnight in a vacuum oven. Break up the pellet by tapping on the tube with the lid closed. Add the SPE column solution into the dried silica microsphere tube and vortex to mix.</li>
 <li>Load 4 &mu;l of the SPE solution into the same channel and then crosslink it by UV irradiation for 2.5 min. Then, flip the chip over and irradiate for another 2.5 min. The polymerized SPE column should be an opaque white solid.</li>
 <li>Wash the channel with 500 &mu;l of 100% methanol to rinse away excess reactants. This wash also removes intra-polymer porogenic solvents, thereby creating the open pore structure of the SPE column when dried in ambient conditions.</li>
 <li>Attach two thin-film heaters to the bottom of the chip with thermally conductive tape. The sides of the heaters need to be aligned with the edge of the wide channels for denaturation and annealing separately (see Figure 1).</li>
 <li>Place five thermocouples into the chip through the dead ended open side channels of each chip (see Figure 2b). The thermocouples stay in these channels via press fit. Tape was used to further fix the location of thermocouples.</li>
</ol>
2. Solid Phase Extraction Method
<ol>
 <li>Prepare 96 &mu;l of 70% ethanol with DEPC-treated and autoclaved water (deionized or Millipore-filtered).</li>
 <li>Add 4 &mu;l of 25X RNA Secure to 96 &mu;l of 70% ethanol in one tube, and duplicate the same mixture with 100% ethanol in a separate tube.</li>
 <li>Prepare 300 &mu;l of channel buffer by mixing the following: 75 &mu;l of 6 M guanidine thiocyanate (GuSCN),150 &mu;l of 2-propanol, 63 &mu;l of nuclease free water, and 12 &mu;l of 25X RNA Secure.</li>
 <li>Prepare 312 &mu;l of lysis buffer by mixing: 100 &mu;l of 6 M GuSCN, 200 &mu;l of 2-propanol, and 12 &mu;l 25X RNA Secure.</li>
 <li>Heat the 70% ethanol, 100% ethanol, channel buffer and lysis buffers at 60 &deg;C for 10-20 min to activate the RNA Secure.</li>
 <li>To equilibrate the microfluidic channel, run the channel buffer through the SPE channel at a flow rate of 0.8 ml/hr.</li>
 <li>Quick-thaw the test sample in VTM at 37 &deg;C in water bath, and remove the sample immediately from water bath as soon as it is thawed.</li>
 <li>Centrifuge sample at 13,000 rpm for 10 min, and transfer 100 &mu;l of the supernatant into 300 &mu;l of the lysis buffer. Complete the mixture by adding 6 &mu;l of 1 &mu;g/&mu;l carrier RNA.</li>
 <li>Vortex to mix, and after spin for 5 sec, load the entire lysate into a Luer-lok 1 cc syringe.</li>
 <li>Run lysate through the SPE channel at a flow rate of 0.8 ml/hr .</li>
 <li>Wash the SPE channel with 100 &mu;l of 70% ethanol , followed by 100 &mu;l of100% ethanol at 1 ml/hr. (There is a 50 &mu;l dead volume in the syringe tip that will not get pushed out, so actually only 50 &mu;l is going through the channel.)</li>
 <li>Position an empty syringe at the 0.5 ml mark and push air through the channel to dry it at 1 ml/hr.</li>
 <li>Run 67.5 &mu;l of nuclease-free water through the channel to elute the bound nucleic acids at 0.5 ml/hr, and collect 13.5 &mu;l from the nanoport at the waste port. (The fluid loss is due to the 54 &mu;l dead volume in the syringe tip and the SPE channel.)</li>
</ol>
3. RT-PCR
<ol>
 <li>Prepare 36.5 &mu;l of the RT-PCR master mix during the air dry step (2.12) using the reagents in a Qiagen OneStep RT-PCR kits.</li>
 <li>Load RT-PCR reagent in the nanoport at the waste port, and mix with RNA to get the following 50 &mu;l RT-PCR reaction: 13.5 &mu;l eluted RNA in nuclease free water, 4 &mu;l RT-PCR enzyme mix, 10 &mu;l Q solution, 10 &mu;l1X one step buffer, 2 &mu;l 25 mM MgCl2, 1 &mu;l 50 &mu;M forward primer 5'-GAC CRA TCC TGT CAC CTC TGA C-3', 1 &mu;l 50 &mu;M reverse primer 5'-AGG GCA TTY TGG ACA AAK CGT CTA-3', 2 &mu;l dNTP, 0.75 &mu;l 1.0% w/v BSA, 0.73 &mu;l PEG8000 and 5.02 &mu;l nuclease free water.</li>
 <li>Load the RT-PCR mixture into the RT channel by gentle vacuum (we used the wall vacuum) and seal the Nanoport with a closed fitting.</li>
 <li>Apply 35 V to heater 1. Keep the reagents in the RT channel for 30 min after the heater 1 equilibrates at 50 &deg;C. Keep the reagents in the RT channel for 15 min after the heater 1 equilibrates at 95 &deg;C.</li>
 <li>Apply 27 V to heater 1 and 50 V to heater 2, wait about three minutes or until heater 1 equilibrates at 60 &deg;C and heater 2 equilibrates at 95 &deg;C.</li>
 <li>Push the reagents into the PCR channel at 0.5 &mu;l /min. It should take about 20 min for the reagents to flow through the serpentine channel.</li>
 <li>As the sample makes it to the end of the serpentine channel, collect the PCR products at the outlet using an appropriately sized pipettor and tip.</li>
</ol>
4. PCR Products Detection
<ol>
 <li>We use an Agilent High Sensitivity DNA test to detect the products. To run this test take out the Agilent High Sensitivity DNA Kit from refrigerator 30 min before testing.</li>
 <li>Turn on the Bioanalyzer and open the 2100 Expert software.</li>
 <li>Load the 1 &mu;l of the PCR products into the testing and follow the Agilent protocol (<a href="http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf" target="_blank">http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf</a>).</li>
 <li>Analyze and record the data.</li>
</ol>

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The authors have declared no competing financial interests.

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

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JoVE Journal

Bioengineering

14.9K Views.  Boston University. Integrato chip microfluidica termoplastico è stato sviluppato per l'utilizzo come diagnostica molecolare. Il chip esegue l'estrazione di acido nucleico, la trascrittasi inversa e PCR. Metodi per la fabbricazione e l'esecuzione del chip sono descritti.

Video: Microfluidic Chip Fabrication and Method to Detect Influenza

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 ...

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

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 ...

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 Fabrication of Polymeric and Biohybrid Fibers with Predesigned 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 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 ...

A Microfluidic Chip for ICPMS Sample Introduction

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 ...

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 ...

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

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 ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

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 ...