We would like to thank NASA Astrobiology Science and Technology Instrument Development (ASTID) Program for funding support (grant numbers 103498 and 103692), Gale Lockwood of the Birck Nantechnology Center at Purdue University for wirebonding of the MAB devices, and Joon Hyeong Park for the CAD drawing of the flow-cell chamber.

1. Preparation of Poly(3,4-ethylenedioxythiophene):Poly(sodium 4-styrenesulfonate) (PEDOT:PSS) Electropolymerization Solution for H+ and CO32- Ions
<ol>
	<li>Add 70 mg poly(sodium 4-styrenesulfonate) (Na+PSS-) to 10 ml deionized (DI) water and vortex until completely dispersed (approx. 10 sec).</li>
	<li>Add 10.7 &mu;l 3,4-ethlyenedioxythiophene (EDOT) to the solution in 1.1 and vortex until solution is completely mixed.</li>
</ol>
2. Preparation of Poly(3,4-ethylenedioxythiophene):Calcium sulphate (PEDOT:CaSO4) Electropolymerization Solution for Ca2+ Ions
<ol>
	<li>Add 136 mg calcium sulphate (CaSO4) to 10 ml DI water and vortex; the solution will not completely disperse and appears milky.</li>
	<li>Add 10.7 &mu;l EDOT to the solution in 2.1 and vortex until completely mixed.</li>
</ol>
3. Electropolymerization of PEDOT-based Conductive Polymer
<ol>
	<li>A Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3) and an EC epsilon potentiostat/galvanostat are used to form the electrochemical cell for electropolymerization. Place the EDOT:PSS electropolymerization solution in the electrochemical cell and nitrogen bubble for 20 min to remove dissolved oxygen.</li>
	<li>Now clip a platinum-gauze at the counter electrode position of the electrochemical cell. Then clip the MAB at the working electrode position of the electrochemical cell with the working electrodes facing the platinum-gauze. Adjust the MAB depth so that only the circular electrodes are submerged in the PEDOT:PSS electropolymerization solution. Avoid solution contact with the square electrical contact pads.</li>
	<li>Place a BASI saturated silver/silver chloride (Ag/AgCl) electrode in the reference electrode position of the electrochemical cell. Make sure that the reference electrode is not in between the working and counter electrodes.</li>
	<li>For PEDOT:PSS deposition: Bubble the electrochemical cell for 20 min, and use the EC epsilon potentiostat/galvanostat to run a single cyclic voltammogram from 0V - 1.1V with a scan rate of 20 mV/sec on a &plusmn;100 &mu;A scale.</li>
	<li>For PEDOT:CaSO4 deposition: Bubble the electrochemical cell for 20 min, and use the EC epsilon potentiostat/galvanostat to run chronopotentiometry at 814 nA for 30 min.</li>
</ol>
4. Cyclic Voltammetry of PEDOT-based Polymer Conjugates in K3Fe(CN)6
<ol>
	<li>Perform steps 3.1- 3.3 above.</li>
	<li>Use the EC epsilon potentiostat/galvanostat to run single cyclic voltammograms from -653 mV to 853 mV with varying scan rates of (25, 50, 75, 100, l25, 150, 175, 200) mV/sec on a &plusmn;10 &mu;A scale.</li>
</ol>
5. Surface Functionalization Protocol
<ol>
	<li>Deposit conductive polymer conjugate specific for the ions of interest as in Step 3.</li>
	<li>Apply ion-selective membrane as in Step 6.</li>
</ol>
6. Application of Ion-selective Membrane
<ol>
	<li>Center the MAB on the vacuum spinner chuck.</li>
	<li>Deposit 100 &mu;l membrane onto the center of the MAB and run.</li>
	<li>Spin-coat ion-selective membrane with a spin coater at 1,500 rpm for 30 sec with a 5 sec ramp up and down.</li>
	<li>Vacuum the spin-coated MAB for 30 min and bake the chip in an oven at 70 &deg;C for 20 min.</li>
</ol>
7. Calibration of PEDOT-PSS Conductive Polymer Conjugate with pH and Carbonate (CO32-) Ion-selective Membrane
<ol>
	<li>Condition the MAB overnight in 10 &mu;M sodium bicarbonate (NaHCO3) and 5 mM potassium chloride (KCl) in algal media.</li>
	<li>Insert the MAB into the microfluidic flow-cell chip holder.</li>
	<li>Inject 5 ml test solution with initial pH value or concentration (e.g. pH 4 or 10 &mu;M for CO32-). Remove bubbles from the flow-cell chip holder.</li>
	<li>Place the flow-cell chip holder onto the flow-cell electrical fixture.</li>
	<li>Open the EC epsilon software and enter open-circuit potential (OP) mode. Set the time to 300 min, the voltage scale to &plusmn; 1V, and the cutoff frequency to 10 kHz, and record the value every 2 sec.</li>
	<li>Let the MAB stabilize (look for a flat line) before continuing with the calibration process.</li>
	<li>Once the MAB is stabilized, flush the flow cell with test solution and inject the next concentration to be calibrated (pH 5 or 25 &mu;M CO32-). Make sure that no bubbles are allowed to enter the flow cell. Repeat steps 7.5 and 7.6 for pH 6, 7, 8, and 9 or CO32- concentrations of 50, 75, 100, 250, 500, 750, and 1,000 &mu;M.</li>
	<li>After the last concentration has run, remove the MAB and dry with nitrogen air.</li>
	<li>Place the MAB back into fresh conditioning solution until next use.</li>
</ol>
8. Calibration of PEDOT:CaSO4 Conductive Polymer Conjugate in CaCl2
<ol>
	<li>Condition the MAB overnight in 7 ml of 0.1 M CaCl2 and 10 &mu;M NaNO3.</li>
	<li>Follow steps similar to 7.2 - 7.10. In step 8.3, replace carbonate test solution with an initial concentration of 0.01 mM CaCl2. Repeat for test-solution concentrations of 0.05, 0.1, 0.5, 1 and 10 mM.</li>
</ol>

<ol>
	<li>Migdalski, J., Bas, B., Blaz, T., Golimowski, J., Lewenstam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Miniaturized+and+Integrated+Galvanic+Cell+for+the+Potentiometric+Measurement+of+Ions+in+Biological+Liquids.">A Miniaturized and Integrated Galvanic Cell for the Potentiometric Measurement of Ions in Biological Liquids.</a> J. Solid State Electrochem. 13, 149-155 (2009).</li><li>Buehler, M. G., Kounaves, S. P., Martin, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+Water-quality+Monitor+with+Ion-selective-electrodes.">Designing a Water-quality Monitor with Ion-selective-electrodes.</a> 1, 331-338 (2001).</li><li>Adamchuk, V. I., Lund, E. D., Sethuramasamyraja, B., Morgan, M. T., Doberman, A., Marx, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Soil+Chemical+Properties+on-the-go+using+Ion-selective-electrodes.">Direct Measurement of Soil Chemical Properties on-the-go using Ion-selective-electrodes.</a> Journal Computers and Electronics in Agriculture. 48 (3), 272-294 (2005).</li><li>Oel&#946;ner, W., Hermann, S., Kaden, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Sensors+and+Sensor+Module+for+Studying+Biological+Systems+in+Space+Vehicles.">Electrochemical Sensors and Sensor Module for Studying Biological Systems in Space Vehicles.</a> Aerospace Science and Technology. 1, 291-296 (1997).</li><li>Bobacka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conducting+Polymer-based+Solid-state+Ion-selective+Electrodes.">Conducting Polymer-based Solid-state Ion-selective Electrodes.</a> Electroanalysis. 18 (1), 7-18 (2006).</li><li>Buck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion Selective Electrodes in Analytical Chemistry. , (1980).</li><li>Nam, H., Cha, G. S., Yang, V. C., Ngo, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18.">Chapter 18.</a> Biosensors and their Applications. , (2000).</li><li>Anatova-Ivanova, S., Mattinen, U., Radu, A., Bobacka, J., Lewenstem, A., Migdalski, J., Danielewski, M., Diamond, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Miniature+All-solid-state+Potentiometric+Sensing+System.">Development of Miniature All-solid-state Potentiometric Sensing System.</a> Sensors and Actuators B. 146, 199-205 (2010).</li><li>Michalska, A., Galuszkiewicz, A., Ogonowska, M., Ocypa, M., Maksymiuk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEDOT+Films:+Multifunctional+Membranes+for+Electrochemical+Ion+sensing.">PEDOT Films: Multifunctional Membranes for Electrochemical Ion sensing.</a> J. Solid State Electrochem. 8, 381-389 (2004).</li><li>Bard, A. J., Faulkner, L. R., ed, 2. n. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electrochemical Methods: Fundamentals and Applications. , (2000).</li><li>Claussen, J. C., Artiles, M. S., McLamore, E. S., Mohanty, S., Shi, J., Rickus, J., Fisher, T. S., Porterfield, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+Glutamate+Biosensing+with+Naanocube+and+Nanosphere+Augmented+Single-walled+Carbon+Nanotube+Networks:+A+Comparative+Study.">Electrochemical Glutamate Biosensing with Naanocube and Nanosphere Augmented Single-walled Carbon Nanotube Networks: A Comparative Study.</a> J. Mater. Chem. 21, 11224-11231 (2011).</li><li>Bobacka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+Stability+of+All-solid-state+Ion-selective+Electrodes+using+Conducting+Polymers+as+Ion-to-electron+Transducers.">Potential Stability of All-solid-state Ion-selective Electrodes using Conducting Polymers as Ion-to-electron Transducers.</a> Anal. Chem. 71, 4932-4937 (1999).</li><li>Lee, J. H., Yoon, I. J., Yoo, C. L., Pyun, H. J., Cha, G. S., Nam, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiometric+Evaluation+of+Solvent+Polymeric+Carbonate-selective+Membranes+based+on+Molecular+Tweezer-type+Neutral+Carriers.">Potentiometric Evaluation of Solvent Polymeric Carbonate-selective Membranes based on Molecular Tweezer-type Neutral Carriers.</a> Anal. Chem. 72, 4694-4699 (2000).</li><li>Song, F., Ha, J., Park, B., Kwak, T. H., Kim, I. T., Nam, H., Cha, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-solid-state+Carbonate+Selective+Electrode+based+on+a+Molecular+Tweezer-type+Neutral+Carrier+with+Solvent-soluble+Conducting+Polymer+Solid+Contact.">All-solid-state Carbonate Selective Electrode based on a Molecular Tweezer-type Neutral Carrier with Solvent-soluble Conducting Polymer Solid Contact.</a> Talanta. 57, 263-270 (2002).</li></ol>

We have nothing to disclose.

The MAB biochip consists of ASSISEs that are constructed from an ISM atop a PEDOT-based CP conjugate transduction layer on a Pt electrode, the combination of which transduces the ionic concentration of interest to a measurable electrical signal. A stable electrode potential is defined by both the CP layer and the ISM layer. Both layers also determine the working lifetime of the MAB and the quality (noise, drift) of the measured electrical signal.
PEDOT is especially attractive as a transductio...

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the items</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments </td>
		 </tr>
		 <tr>
			<td>3,4-Ethylenedioxythiophene</td>
			<td>Sigma-Aldrich</td>
			<td>483028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly(sodium 4-styrenesulfonate)</td>
			<td>Sigma-Aldrich</td>
			<td>243051</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EC epsilon galvanostat/potentiostat</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>e2P</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Saturated Ag/AgCl reference electrode</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>MF-2052</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pt gauze</td>
			<td>Alfa Aesar</td>
			<td>10283</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P-8131</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium nitrate</td>
			<td>J.T. Baker</td>
			<td>3190-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate</td>
			<td>Mallinckrodt/ Macron</td>
			<td>7412-12</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S-7127</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium chloride</td>
			<td>J.T. Baker</td>
			<td>1311-01</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>237132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C3 cell stand</td>
			<td>Bioanalytical Systems Inc.</td>
			<td>EF-1085</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flow-cell chip holder</td>
			<td></td>
			<td></td>
			<td>Custom, courtesy of NASA Ames</td>
		 </tr>
		 <tr>
			<td>Flow-cell electrical fixture</td>
			<td></td>
			<td></td>
			<td>Custom, courtesy of NASA Ames </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Specific reagents and equipment.</td>
		 </tr>
		</tbody>
 </table>

multi-analyte biochip (mab) based on all-solid-state ion-selective electrodes (assise) for physiological research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media 1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H+, Ca2+, and CO32- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.
The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface 5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) 7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).
Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO32- (measured range 0.01 mM - 1 mM), and Ca2+ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.
The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.
Biochip Design and Experimental Methods
The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 &mu;m in diameter and is equally spaced at 1.4 mm from the REs, which are 480 &mu;m in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.
Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H+ and CO32-, while one with sulphate (added to the solution as CaSO4) is utilized for Ca2+. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area 10. Spin-coating at 1,500 rpm is used to cast ~2 &mu;m thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).
The MAB is contained in a microfluidic flow-cell chamber filled with a 150 &mu;l volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

An example of a cyclic voltammogram (CV) result of PEDOT:PSS and its corresponding cathodic peak current (ip) vs. the scan rate (v1/2) are shown in Figures 5a and 5b respectively. PEDOT:CaSO4 at various scan rates and its cathodic peak current are not shown. Using Randles-Sevcik analysis 10, the effective surface areas of the solid contact PEDOT:PSS and PEDOT:CaSO4 without ion-selective membrane were found to be 4.4...

Watch this Scientific Journal Video about Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research at JoVE.com

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

multi-analyte-biochip-mab-based-on-all-solid-state-ion-selective

Research

JoVE Journal

Bioengineering

17.1K Views.  Purdue University. All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

Video: Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

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 ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

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 ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the dynamic properties of the cytoskeleton. This protocol offers a technique for human primary fibroblast transfection, which can be difficult because of the specifics of primary cell cultivation conditions. Additionally, cytoskeleton dynamic property maintenance requires a low level of transfection to obtain a good signal-to-noise ratio without causing microtubule stabilization. It is important to take measures to protect the cells from light-induced stress and fluorescent dye fading. In the course of our work, we tested different transfection methods and protocols as well as different vectors to select the best combination of conditions suitable for human primary fibroblast studies. We analyzed the resulting time-lapse videos and calculated microtubule dynamics using ImageJ. The dynamics of microtubules' plus-ends in the different cell parts are not similar, so we divided the analysis into subgroups - the centrosome region, the lamella, and the tail of fibroblasts. Notably, this protocol can be used for in vitro analysis of cytoskeleton dynamics in patient samples, enabling the next step towards understanding the dynamics of the various disease development.

Microtubule Plus-End Dynamics Visualization in Huntington&#039;s Disease Model based on Human Primary Skin Fibroblasts

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington's disease patients.

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...