The authors acknowledge the Office of Naval Research, United Soybean Board, and National Science Foundation for funding.

1. Modification of Nafion with Quaternary Ammonium Salts
<ol>
	<li>Shake a bottle of 5% w/v Nafion suspension vigorously for approx. 30 seconds to ensure that Nafion is suspended uniformly in solution.</li>
	<li>Pipette out 2 mL of the now re-suspended Nafion into a glass vial (vial volume could contain from 2.5 mL to 10 mL).</li>
	<li>Measure a 3-fold molar excess (relative to the sulfonic acid groups on the Nafion polymer) of the alkyl ammonium bromide salt (appropriate masses are shown in Table 1) and add this to the vial containing the 2 mL of Nafion.</li>
	<li>Vortex the vial at 1500 rpm for 10-15 minutes.</li>
	<li>Pour the viscous solution into a plastic weighing tray that measures approx. 3 x 3 in., and use a pipette to transfer any residual solution from the vial to the weighing tray.</li>
	<li>Allow the solvents to evaporate out of the weigh boat, leaving a yellow/brown, transparent film on the bottom of the weighing tray (Figure 2). The rate of solvent evaporation should be such that it takes more than 6 hours for all solvent to evaporate. If the solvent evaporates too fast, a white, crusty material will form instead of a transparent film indicating that the micellar structure of the polymer has been destroyed, and you must re-start the procedure. If solvent evaporation is too slow, a de-humidifier may be necessary, because too slow of evaporation typically leads to a tacky, orange gel and you must re-start the procedure. Typical temperature ranges for solvent evaporation are 20 - 37 &deg;C. The actual conditions for drying are a function of the relative humidity and temperature in the room, but it is important that drying being slow to maintain micelle structure, but not too slow to allow for gel formation.</li>
	<li>Fill the weigh boat with 18M &Omega;cm de-ionized water (10-20 mL of water), cover, and allow to soak for 12-24 hours to remove excess alkyl ammonium bromide salts and HBr.</li>
	<li>Pour (or pipette) out the water and rinse 3 times with enough DI water to fill the tray each time. Be careful not to lose any of the polymer film during this step.</li>
	<li>Allow the weighing tray to sit uncovered until the polymer is completely dry At this point, the polymer should be a clear and somewhat brittle plastic film. Again, if the air is very humid, a de-humidifier may be necessary to complete the evaporation in a timely manner.</li>
	<li>Using a spatula, carefully remove the dried film from the weighing tray and transfer it into a clean glass vial.</li>
	<li>Add 2 mL of ethanol and 3 ceramic mixing beads, and vortex for 4 hours or until the polymer film is completely re-suspended.</li>
</ol>
2. Immobilization of Enzymes into TBAB-Modified Nafion for Activity Assays
<ol>
	<li>For a dry enzyme, weigh out 1-10 mg of the enzyme into a 1.5 mL microcentrifuge tube, and add 1 mL of 100 mM pH 7 phosphate buffer to create a 1-10 mg/mL enzyme solution. For an enzyme that is in solution, use a bicinchoninic acid (BCA) assay13 to determine the amount of protein, and add an appropriate amount of 100 mM phosphate buffer to bring the protein concentration to 1-10 mg/mL. 1-10 mg/mL typically corresponds to 1-50 nanomoles/mL.</li>
	<li>To 120 &mu;L of the 1 mg/mL enzyme solution, add 60 &mu;L of the alkyl ammonium-modified Nafion solution, and vortex for 10 seconds. (This mixture can be scaled up for large numbers of replicates. Keep the enzyme-to-polymer solution ratio at 2:1.)</li>
	<li>Pipette 60 &mu;L of the enzyme/polymer solution into the bottom of 3 separate 1 cm2 cuvettes, and allow to dry overnight.</li>
</ol>
3. Assay of Immobilized NAD-Dependent Dehydrogenase Enzyme
<ol>
	<li>To the cuvette, add 1.3 mL of 50 mM sodium pyrophosphate (pH 8.8), 1.5 mL of 15 mM NAD (freshly prepared), and 0.1 mL water.</li>
	<li>Place the cuvette in a UV/Vis spectrophotometer (i.e. ThermoScientific Evolution 260 Bio and Thermo Spectronic Genesys 20) set to a wavelength of 340 nm.</li>
	<li>Zero the spectrometer, and then add 0.1 mL ethanol. Mix the reagents by gently pipetting the solution up and down 5 times. For a blank, use 0.1 mL of additional water instead of the 0.1 mL of ethanol.</li>
	<li>Record the absorbance at 340 nm at 5 minutes after the reagents were added to the cuvette and 20 minutes after. Plot the two data points to get a slope that can be used for activity calculations.</li>
</ol>
4. Assay of Immobilized PQQ-Dependent Dehydrogenases
<ol>
	<li>To the cuvette, add 1.5 mL of sodium phosphate (pH 7.3) and 200 &mu;L of 600 &mu;M PMS.</li>
	<li>Place the cuvette in a UV/Vis spectrophotometer set to a wavelength of 600 nm and then zero the spectrometer.</li>
	<li>Add 100 &mu;L of 700 &mu;L DCIP and 200 &mu;L of the substrate of interest (ethanol, acetaldehyde, glycerol, glucose, or glyceraldehyde), and mix the reagents by gently pipetting the solution up and down 5 times. For a blank, use 200 &mu;L of water instead of the substrate of interest.</li>
	<li>Record the absorbance at 600 nm at 5 minutes after the reagents were added to the cuvette and 20 minutes after.</li>
</ol>
5. Assay of Immobilized Glucose Oxidase
<ol>
	<li>To the cuvette, add 2.0 mL of a solution containing 0.2 M p-hydroxybenzoic acid, 0.02 % (w/v) sodium azide, 128 U peroxidase, 0.3 mM 4-aminoantipyrine, 1 M potassium phosphate, and 50 mM glucose. Mix the solution by pipetting up and down 5 times.</li>
	<li>Place the cuvette in a UV/Vis spectrophotometer set to a wavelength of 510 nm.</li>
	<li>Record the absorbance at 510 nm at 5 minutes after the reagents were added to the cuvette and again at 20 minutes after.</li>
</ol>
6. Representative Results
The micellar structure of the modified Nafion polymer can be disrupted by drying the original salt/polymer co-casted film too fast. Figure 2 shows a salt/polymer mixture that has been dried correctly resulting in a transparent, light brown film. A film that dries too fast can result in opaque, white flakes of polymer due to the fact that the drying process can destroy the micellar structure.
Once the modified Nafion polymer and enzyme have been mixed and co-cast onto the bottom of a cuvette, enzymatic activity assays can be used to assess the stability of the enzyme within the polymer film. Tables 2-4 show assay results of two dehydrogenase enzymes and glucose oxidase immobilized into various modified Nafion films, respectively. Note the higher activity of the enzymes that are immobilized vs. the enzymes in buffer solution, showing that modified Nafion polymers can actually enhance the activity of certain enzymes (called superactivity). Other enzymes have transport limitations that decrease their specific activity when immobilize them in the polymer (i.e. cellulases and amylases, whose substrates are quite large macromolecules).
<table border="1">
	<tbody>
		<tr>
			<td>Quaternary ammonium salt used</td>
			<td>3 fold excess</td>
		</tr>
		<tr>
			<td>T3A (tetrapropylammonium bromide)</td>
			<td>32.37 mg/ml</td>
		</tr>
		<tr>
			<td>TBAB (tetrabutylammonium bromide)</td>
			<td>39.19 mg/ml</td>
		</tr>
		<tr>
			<td>TPAB (tetrapentylammonium bromide)</td>
			<td>46.01 mg/ml</td>
		</tr>
		<tr>
			<td>TEHA (triethylhexylammonium bromide)</td>
			<td>32.37 mg/ml</td>
		</tr>
		<tr>
			<td>TMHA (trimethylhexylammonium bromide)</td>
			<td>27.25 mg/ml</td>
		</tr>
		<tr>
			<td>TMOA (trimethyloctylammonium bromide)</td>
			<td>30.66 mg/ml</td>
		</tr>
		<tr>
			<td>TMDA (trimethyldecylammonium bromide)</td>
			<td>34.07 mg/ml</td>
		</tr>
		<tr>
			<td>TMDDA (trimethyldodecylammonium bromide)</td>
			<td>37.48 mg/ml</td>
		</tr>
		<tr>
			<td>TMTDA (trimethyltetradecylammonium bromide)</td>
			<td>40.89 mg/ml</td>
		</tr>
		<tr>
			<td>TMHDA (trimethylhexadecylammonium bromide)</td>
			<td>44.31 mg/ml</td>
		</tr>
		<tr>
			<td>TMODA (trimethyloctadecylammonium bromide)</td>
			<td>47.71 mg/ml</td>
		</tr>
	</tbody>
</table>
Table 1. Amounts of tetra-alkyl ammonium salts to use for Nafion polymer modification.
<table border="1">
	<tbody>
		<tr>
			<td>Type of Nafion </td>
			<td>Enzyme activity (U/g)</td>
		</tr>
		<tr>
			<td>Buffer (no polymer)</td>
			<td>16.63 &plusmn; 8.11</td>
		</tr>
		<tr>
			<td>Nafion (un-mod.)</td>
			<td>9.25 &plusmn; 2.21</td>
		</tr>
		<tr>
			<td>TMTDA</td>
			<td>3.23 &plusmn; 2.92</td>
		</tr>
		<tr>
			<td>TBAB</td>
			<td>3.93 &plusmn; 3.33</td>
		</tr>
		<tr>
			<td>TMDDA</td>
			<td>4.19 &plusmn; 1.04</td>
		</tr>
		<tr>
			<td>TMOA</td>
			<td>3.51 &plusmn; 1.11</td>
		</tr>
		<tr>
			<td>TMDA</td>
			<td>8.00 &plusmn; 4.53</td>
		</tr>
		<tr>
			<td>TMHA</td>
			<td>1.68 &plusmn; 1.39</td>
		</tr>
		<tr>
			<td>TMHDA</td>
			<td>4.83 &plusmn; 0.99</td>
		</tr>
		<tr>
			<td>TMODA</td>
			<td>10.45 &plusmn; 3.20</td>
		</tr>
	</tbody>
</table>
Table 2. NAD-dependent glucose dehydrogenase activity immobilized in selected modified Nafion polymers (note: immobilized activity is a function of initial specific activity of the enzyme).
<table border="1">
	<tbody>
		<tr>
			<td>Type of Nafion </td>
			<td>Enzyme activity (mU/g)</td>
		</tr>
		<tr>
			<td>Buffer (no polymer)</td>
			<td>7.18 &plusmn; 0.51</td>
		</tr>
		<tr>
			<td>Nafion (un-mod.)</td>
			<td>70.1&plusmn; 0.5</td>
		</tr>
		<tr>
			<td>TMTDA</td>
			<td>133 &plusmn; 6</td>
		</tr>
		<tr>
			<td>TBAB</td>
			<td>244 &plusmn; 4</td>
		</tr>
		<tr>
			<td>TMDDA</td>
			<td>221 &plusmn; 6</td>
		</tr>
		<tr>
			<td>TMOA</td>
			<td>1.78 &plusmn; 0.63</td>
		</tr>
		<tr>
			<td>TMDA</td>
			<td>206 &plusmn; 5</td>
		</tr>
		<tr>
			<td>TEHA</td>
			<td>40.1 &plusmn; 50.6</td>
		</tr>
		<tr>
			<td>TMHDA</td>
			<td>0</td>
		</tr>
		<tr>
			<td>TMODA</td>
			<td>1.45 &plusmn; 0.06</td>
		</tr>
	</tbody>
</table>
Table 3. PQQ-dependent glucose dehydrogenase activity immobilized in selected modified Nafion polymers (note: immobilized activity is a function of initial specific activity of the enzyme).
<table border="1">
	<tbody>
		<tr>
			<td>Type of Nafion </td>
			<td>Enzyme activity (U/g)</td>
		</tr>
		<tr>
			<td>Buffer (no polymer)</td>
			<td>103.61 &plusmn; 3.15</td>
		</tr>
		<tr>
			<td>Nafion (un-mod.)</td>
			<td>19.93 &plusmn; 10.10</td>
		</tr>
		<tr>
			<td>TMTDA</td>
			<td>247.25 &plusmn; 12.49</td>
		</tr>
		<tr>
			<td>TBAB</td>
			<td>152.27 &plusmn; 5.29</td>
		</tr>
		<tr>
			<td>TMDDA</td>
			<td>262.05 &plusmn; 6.26</td>
		</tr>
		<tr>
			<td>TMOA</td>
			<td>129.18 &plusmn; 2.31</td>
		</tr>
		<tr>
			<td>TMDA</td>
			<td>141.23 &plusmn; 1.97</td>
		</tr>
		<tr>
			<td>TMHA</td>
			<td>131.75 &plusmn; 2.89</td>
		</tr>
		<tr>
			<td>TMHDA</td>
			<td>132.50 &plusmn; 1.18</td>
		</tr>
		<tr>
			<td>TMODA</td>
			<td>136.50 &plusmn; 0.96</td>
		</tr>
	</tbody>
</table>
Table 4. Representative glucose oxidase specific activity immobilized in selected modified Nafion polymers (note: immobilized activity is a function of initial specific activity of the enzyme).
<img alt="Figure 1" src="/files/ftp_upload/3949/3949fig1.jpg" /> 
Figure 1. Schematic of TBAB incorporation into Nafion polymer and subsequent use in enzyme immobilization.
<img alt="Figure 2" src="/files/ftp_upload/3949/3949fig2.jpg" /> 
Figure 2. Optical photograph of initial co-cast films of Nafion and TBAB. Slow drying yields a transparent, light brown film covering the bottom of the weighing tray.

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	<li>Calabrese-Barton, S., Gallaway, J., Atanassov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+biofuel+cells+for+implantable+and+microscale+devices+Chem.">Enzymatic biofuel cells for implantable and microscale devices Chem.</a> Rev. 104, 4867-4886 (2004).</li><li>Cracknell, J. A., Vincent, K. A., Armstrong, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+as+Working+or+Inspirational+Electrocatalysts+for+Fuel+Cells+and+Electrolysis.">Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis.</a> Chem. Rev. 108, 2439-2461 (2008).</li><li>Minteer, S. D., Liaw, B. Y., Cooney, M. 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No conflicts of interest declared.

In the described procedure, tetra-alkyl ammonium salts are used to modify commercial Nafion to create micellar polymers that can be used to immobilize and stabilize enzymes. The assays described in the procedure show that the polymer can be used to immobilize a wide variety of enzymes with a high retention of activity. If the enzyme of interest has very low activity or is impure, a higher concentration may be required and should not affect the immobilization process, unless immobilizing enzymes in concentrations ...

<table border="1">
		<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td>Nafion</td>
 <td>Sigma-Aldrich</td>
 <td>70160</td>
 </tr>
 <tr>
 <td>Tetra alkylammonium bromide salts</td>
 <td>Sigma-Aldrich</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>Alcohol dehydrogenase</td>
 <td>Sigma-Aldrich</td>
 <td>A3263</td>
 </tr>
 <tr>
 <td>Nicotinamide adenine dinucleotide (NAD)</td>
 <td>Simga-Aldrich</td>
 <td>N7004</td>
 </tr>
 <tr>
 <td>Sodium pyrophosphate</td>
 <td>Sigma-Aldrich</td>
 <td>P8010</td>
 </tr>
 <tr>
 <td>Phenazine methosulfate (PMS)</td>
 <td>Sigma-Aldrich</td>
 <td>P9625</td>
 </tr>
 <tr>
 <td>2,6-Dichloroindophenol (DCIP)</td>
 <td>Sigma-Aldrich</td>
 <td>D1878</td>
 </tr>
 <tr>
 <td>Glucose oxidase</td>
 <td>Sigma-Aldrich</td>
 <td>G7141</td>
 </tr>
 <tr>
 <td>4-Hydroxybenzoic acid</td>
 <td>Sigma-Aldrich</td>
 <td>240141</td>
 </tr>
 <tr>
 <td>Sodium azide</td>
 <td>Sigma-Aldrich</td>
 <td>S8032</td>
 </tr>
 <tr>
 <td>Peroxidase</td>
 <td>Sigma-Aldrich</td>
 <td>P8375</td>
 </tr>
 <tr>
 <td>4-aminoantipyrine</td>
 <td>Sigma-Aldrich</td>
 <td>06800</td>
 </tr>
 <tr>
 <td>UV/Vis Spectrophotometer</td>
 <td>Thermo</td>
 <td>Evolution 260 Bio or Spectronic Genesys 20</td>
 </tr>
 <tr>
 <td>Vortex Genie</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Analytical balance</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
	 </tbody>
 </table>

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

Watch this Scientific Journal Video about Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization at JoVE.com

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

hydrophobic-salt-modified-nafion-for-enzyme-immobilization

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16.1K Views.  University of Utah. This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Video: Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...