Name: immobilization of live caenorhabditis elegans individuals using an ultra-thin polydimethylsiloxane microfluidic chip with water retention
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Description: Watch this Scientific Journal Video about Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention at JoVE.com

The authors thank Dr. Atsushi Higashitani for kind advice regarding treatment of C. elegans and Drs. Yuya Hattori, Yuichiro Yokota, and Yasuhiko Kobayashi for valuable discussions. The authors thank the Caenorhabditis Genetic Center for providing strains of C. elegans and E. coli. We thank the crew of the cyclotron of TIARA at QST-Takasaki for their kind assistance with the irradiation experiments. We thank Dr. Susan Furness for editing a draft of this manuscript. This study was supported in part by KAKENHI (Grant Numbers JP15K11921 and JP18K18839) from JSPS to M.S.

1. Strains and maintenance
<ol>
	<li>Select a suitable strain of C. elegans and Escherichia coli (food) depending on the purpose of the experiment. 
	NOTE: In the present paper, wild-type N210C. elegans (Figure 2A) is generally used, and HBR4:goeIs3[pmyo-3::GCamP3.35::unc-54 - 3&#39;utr, unc-119(+)]V11 is only employed for imaging assay. E. coli OP50 was used as food for C...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+activity+of+neurons+and+muscles.">Imaging the activity of neurons and muscles.</a> WormBook. 2, 1-13 (2006).</li><li>Aubry, G., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+optical+developments+in+microfluidic+platforms+for+Caenorhabditis+elegans+research.">A perspective on optical developments in microfluidic platforms for Caenorhabditis elegans research.</a> Biomicrofluidics. 8, 011301 (2014).</li><li>Lumirror Catalog. 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On-chip immobilization of C. elegans under live conditions using a wettable PDMS microfluidic chip enables the efficient targeted microbeam irradiation of multiple animals. The ease of handling and features to prevent drying make this system suitable for applications not only in microbeam irradiation, but also in several behavioral assays. These worm sheets have already been commercialized and can be easily obtained. Conventional microfluidic chips, such as olfactory chips, are associated with problems including...

Radiation, including X-rays, gamma rays, and heavy-ion beam, is widely used for biological applications such as in cancer diagnosis and treatment, and for ion-beam breeding. Numerous studies and technical developments are currently focusing on the effects of radiation1,2,3. Microbeam irradiation is a powerful means of identifying radiosensitive sites in living organisms4. The Takasaki Advanced Radiation Research Institute of National Institutes for Quantum and Radiological Science and Technology (QST-Takasaki) has been developing a technology to irradiate individual cells under microscopic observation using heavy-ion microbeams5, and has established methods to enable targeted microbeam irradiation of several model animals, such as the nematode Caenorhabditis elegans4,6, silkworms7, and Oryzias latipes (Japanese medaka)8. Targeted microbeam irradiation of the nematode C. elegans allows the effective knockdown of specific regions, such as the nerve ring in the head region, thus helping to identify the roles of these systems in processes such as locomotion.
A method for on-chip immobilization of C. elegans individuals without the need for anesthesia has been developed to allow for microbeam irradiation4. In addition, to improve microfluidic chips used in the previous study4, we have recently developed wettable, ion-penetrable, polydimethylsiloxane (PDMS) microfluidic chips, referred to as worm sheets (see Table of Materials), for immobilizing C. elegans individuals9. These comprise of ultra-thin soft sheets (thickness = 300 &#181;m; width = 15 mm; length = 15 mm) with multiple (20 or 25) straight microfluidic channels (depth = 70 &#181;m; width = 60 &#181;m or 50 &#181;m; length = 8 mm) at the surface (Figure 1A-D). The microfluidic channels are open and allow multiple animals to be enclosed in them simultaneously (Figure 1E). The sheets are resilient to allow the microfluidic channels to be expanded (by ~10%, Figure 1F), and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals.
The channels do not hurt the worms when they are being enclosed or when they are collected. Furthermore, the sheets are made from PDMS, which is essentially hydrophobic, but water retention can be achieved by imparting hydrophilicity to the material. The water retention and thickness are favorable characteristics of the worm sheets. The water-retention capacity prevents dehydration of the animals after prolonged immobilization and enables long-term observations to be carried out.
In addition, as described previously9, the sheets are only 300 &#181;m thick, allowing heavy ions such as carbon ions (with a range of about 1 mm in water) to pass through the sheet enclosing the animals. This allows the ion particles to be detected and the applied radiation dose to be measured accurately. Moreover, the worm sheets can be reused and are thus economical. With the conventional injection method, the animals enclosed are sometimes dead and they cannot be taken out of the channel; their eggs can also clog the channels. This makes the chip unusable. Chips are, therefore, basically disposable and the cost-benefit ratio is poor.
In the present paper, we describe in detail a series of methods for on-chip immobilization of live C. elegans individuals using worm sheets. Through locomotion assays of animals 3 h after on-chip immobilization, we evaluated the suitable cover film. In addition, we showed the examples of on-chip immobilization for both imaging observations and microbeam irradiation.

<table border="0" cellpadding="0" cellspacing="0" style="width:1151px;" width="1152">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">C. elegans wild-type strain</td>
			<td style="width:125px;">Caenorhabditis Genetics Center (CGC) , Minnesota, USA</td>
			<td style="width:353px;">N2</td>
			<td style="width:449px;">Wild-type C. elegans strain generally used in this study</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">C. elegans unc-119(e2498) III mutant strain</td>
			<td style="width:125px;">Caenorhabditis Genetics Center (CGC) , Minnesota, USA</td>
			<td style="width:353px;">CB4845</td>
			<td style="width:449px;">C. elegans strain only employed as an example of mutants with abnormal body shape&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:223px;">C. elegans transgenic strain HBR4</td>
			<td style="width:125px;">Caenorhabditis Genetics Center (CGC) , Minnesota, USA</td>
			<td style="width:353px;">HBR4</td>
			<td style="width:449px;">The genotype of this transgenic C. elegans strain is HBR4:goeIs3[pmyo-3::GCamP3.35:: unc-54&#8211;3&#8217;utr, unc-119(+)]V. This strain was only employed for imaging observation.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">E. coli strain</td>
			<td style="width:125px;">Caenorhabditis Genetics Center (CGC) , Minnesota, USA</td>
			<td style="width:353px;">OP50</td>
			<td style="width:449px;">E. coli strain used as food for C. elegans</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Worm Sheet IR (50/60)</td>
			<td style="width:125px;">Biocosm, Inc., Hyogo, Japan</td>
			<td>BCM17-0001</td>
			<td style="width:449px;">Microfluidic chip with 25 straight 50/60-&#181;m width channels used in all experiments and observation in this paper&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Worm Sheet 60</td>
			<td style="width:125px;">Biocosm, Inc., Hyogo, Japan</td>
			<td>BCM18-0001</td>
			<td style="width:449px;">Microfluidic chip with 20 straight 60 &#181;m-width channels. This is sitable for adults 3-5 days after hatching at 20&#176;C.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Worm Sheet 50</td>
			<td style="width:125px;">Biocosm, Inc., Hyogo, Japan</td>
			<td>BCM18-0002</td>
			<td style="width:449px;">Microfluidic chip with 20 straight 50 &#181;m-width channels. This is sitable for youg adults ~3 days after hatching at 20&#176;C.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MICRO COVER GLASS</td>
			<td style="width:125px;">MATSUNAMI GLASS IND. LTD.</td>
			<td>C030401</td>
			<td style="width:449px;">Cover glass (thickness: 130-170 &#181;m) used in locomotion assays in Protocol 3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polystyrene Film</td>
			<td style="width:125px;">Biocosm, Inc., Hyogo, Japan</td>
			<td>BCM18-0001/ BCM18-0002</td>
			<td style="width:449px;">Bundled items of Worm Sheets. PS filim (thickness: ~130 &#181;m) used in locomotion assays in Protocol 3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polyester Film Lumirror</td>
			<td style="width:125px;">TORAY INDUSTRIES, INC., Tokyo, Japan</td>
			<td>Lumirror T60 (t 125 &#181;m)</td>
			<td style="width:449px;">PET filim (thickness: 125 &#181;m) used in locomotion assays in Protocol 3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IWAKI 60 mm/non-treated dish</td>
			<td style="width:125px;">AGC Techno Glass Co., Ltd., Shizuoka, Japan).</td>
			<td>1010-060</td>
			<td style="width:449px;">Non-treated dish used in incuvation of C. elegans in Protocol 1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IWAKI 35 mm/non-treated dish</td>
			<td style="width:125px;">AGC Techno Glass Co., Ltd., Shizuoka, Japan).</td>
			<td>1010-035</td>
			<td style="width:449px;">Non-treated dish used in locomotion assays in Protocol 3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Milli-Q</td>
			<td style="width:125px;">Merck, France</td>
			<td></td>
			<td style="width:449px;">Ultrapure water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kimwipe S-200</td>
			<td style="width:125px;">Nippon Paper Crecia Co., Ltd., Tokyo, Japan</td>
			<td>62020</td>
			<td style="width:449px;">120 mm x 215 mm; 200 sheets/ box</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">WormStuff Worm Pick</td>
			<td style="width:125px;">Genesee Scientific Corporation, CA, USA)</td>
			<td>59-AWP</td>
			<td style="width:449px;">Platina picker specilized for picking up C. elegans</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Research Stereo Microscope System</td>
			<td style="width:125px;">OLYMPUS CORPORATION, Tokyo, Japan</td>
			<td>SZX16</td>
			<td style="width:449px;">Micriscope used in all experiments and observation in this paper</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Motorized Focus Stand for SZX16</td>
			<td style="width:125px;">OLYMPUS CORPORATION, Tokyo, Japan</td>
			<td style="width:353px;">SZX2-ILLB</td>
			<td style="width:449px;">This was used for bright field observation in Protocol 3-8.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Lens (&#215;1)</td>
			<td style="width:125px;">OLYMPUS CORPORATION, Tokyo, Japan</td>
			<td>SDFPLAPO1&#215;PF</td>
			<td style="width:449px;">NA: 0.15; W.D.: 60 mm. This lends was used for bright field observation in Protocol 3-8.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective Lens (&#215;2)</td>
			<td style="width:125px;">OLYMPUS CORPORATION, Tokyo, Japan</td>
			<td>SDFPLAPO2XPFC</td>
			<td style="width:449px;">NA: 0.3; W.D.: 20 mm. This&#160; lends was used for imaging observations.</td>
		</tr>
	</tbody>
</table>

immobilization of live caenorhabditis elegans individuals using an ultra-thin polydimethylsiloxane microfluidic chip with water retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Active C. elegans individuals could be immobilized successfully using an ultra-thin, wettable PDMS, microfluidic chip (worm sheet). We investigated the suitability of different cover films for sealing the worm sheet, as described in protocol section 3. To evaluate the sealing effects of the cover films, we determined the motility of animals 3 h after on-chip immobilization using cover glass (thickness: 130-170 &#181;m), PET film (thickness: 125 &#181;m), and PS film (thickness: ~...

Watch this Scientific Journal Video about Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention at JoVE.com

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

One advantage of this protocol is that the worm sheet is easy to handle. The sheet has features that prevent drying, enabling us to conduct microbeam irradiation to living animals, perform several behavioral assays, and long-term observation. The microfluidic channels in this worm sheet are open, making it easier to collect animals.These worm sheets can also be used repeatedly, making them more economical. When we were developing this technique, we wanted to irradiate only a part of the animals with a microbeam under a microscope. So we needed clear, very thin chips capable of holding the animals.To begin, place a thin, transparent sheet onto the stage for use as a bottom cover film. Gently position a worm sheet on the bottom cover film with flat tweezers. Place at least three droplets of buffer solution on the surface of a 6 centimeter, non-treated Petri dish.Then, pick several adult C.elegans with a platina picker from a culture plate. Transfer the animals to a droplet with the platina picker and allow the animals to swim in buffer to remove any bacteria. Wash the animals in two separate droplets and rinse the animals in another droplet.Next, place a droplet of buffer solution onto the surface of the worm sheet. Remove the washed animals from the buffer droplet, and transfer them to a droplet on the worm sheet. Use flat tweezers to place a PS cover film over the worm sheet, and press gently over the channels from one end of the sheet to the other.Or simply drop animals into droplet and gently seal with the cover film so droplet spreads between the cover and the worm sheets with the animals within the channel. Confirm the animals are alive by checking for movement with a microscope at 1-2x magnification. Record the animals'positions and specifically note the number of the channel in which each animal's enclosed, and the position of each animal in the channel.For collecting immobilizing animals, use flat tweezers to remove the cover film from the worm sheet, then drop 10-15 microliters of buffer solution onto one of the microfluidic channels enclosing the animals. Under the stereomicroscope, observe the animals as they start to spin. Then use the platina picker to pick up the swimming animals and transfer them to an assay plate.After enclosing the animals as previously demonstrated, observe fluorescent spots on the animals with a flourescence microscope. Then image calcium ion wave propagation using video acquisition to observe dynamic activities. Place the used worm sheet onto a six centimeter petri-dish and place about 100 microliters of sterilized ultra-pure water onto the sheet.Use gloved fingers to spread water on the surface of the sheet and wash off any contaminants. Next, use disposable wipes to remove the moisture from the worm sheet. Dispense five milliliters of 70%ethanol into the Petri-dish and use gloved fingers to spread the ethanol over the worm sheet.Finally, after the sheet is dry, place the sheet on a sterile Petri-dish and cover. Using this protocol, the suitability of different cover films was investigated. There was no significant difference in motility between control animals and animals enclosed in microfluidic channels with PS film.In contrast, motility was significantly reduced in animals enclosed under a cover glass. The motility of animals enclosed with a pet film was also significantly decreased. It is important to select cover films for the sealing of microfluidic channels carefully.Using this protocol, we evaluated the suitability of different cover films through locomotion assays of the animals three hours after on-chip immobilization. Using this technique, we intended to develop an ultra-thin wettable chip only for microbeam irradiation. However, these worm sheets have already been used for imaging and behavioral assays beyond their original applications.

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Research

JoVE Journal

Bioengineering

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-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

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.

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 ...

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 ...

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 ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.