Name: Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization (Video) | JoVE Journal
Uploaded: 2012-07-11T12:00:00.000+00:00
Duration: 11 min 16 s
Description: Learn to synthesize hydrophobic salt-modified Nafion for enzyme immobilization and stabilization. This method enhances enzyme stability during activity assays using ammonium salt exchange and controlled drying techniques.

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5 minutes

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 °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 Ωcm 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 μL of the 1 mg/mL enzyme solution, add 60 μL 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 μL 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 μL of 600 μM 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 μL of 700 μL DCIP and 200 μL 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 μL 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 ± 8.11</td>
		</tr>
		<tr>
			<td>Nafion (un-mod.)</td>
			<td>9.25 ± 2.21</td>
		</tr>
		<tr>
			<td>TMTDA</td>
			<td>3.23 ± 2.92</td>
		</tr>
		<tr>
			<td>TBAB</td>
			<td>3.93 ± 3.33</td>
		</tr>
		<tr>
			<td>TMDDA</td>
			<td>4.19 ± 1.04</td>
		</tr>
		<tr>
			<td>TMOA</td>
			<td>3.51 ± 1.11</td>
		</tr>
		<tr>
			<td>TMDA</td>
			<td>8.00 ± 4.53</td>
		</tr>
		<tr>
			<td>TMHA</td>
			<td>1.68 ± 1.39</td>
		</tr>
		<tr>
			<td>TMHDA</td>
			<td>4.83 ± 0.99</td>
		</tr>
		<tr>
			<td>TMODA</td>
			<td>10.45 ± 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 ± 0.51</td>
		</tr>
		<tr>
			<td>Nafion (un-mod.)</td>
			<td>70.1± 0.5</td>
		</tr>
		<tr>
			<td>TMTDA</td>
			<td>133 ± 6</td>
		</tr>
		<tr>
			<td>TBAB</td>
			<td>244 ± 4</td>
		</tr>
		<tr>
			<td>TMDDA</td>
			<td>221 ± 6</td>
		</tr>
		<tr>
			<td>TMOA</td>
			<td>1.78 ± 0.63</td>
		</tr>
		<tr>
			<td>TMDA</td>
			<td>206 ± 5</td>
		</tr>
		<tr>
			<td>TEHA</td>
			<td>40.1 ± 50.6</td>
		</tr>
		<tr>
			<td>TMHDA</td>
			<td>0</td>
		</tr>
		<tr>
			<td>TMODA</td>
			<td>1.45 ± 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 ± 3.15</td>
		</tr>
		<tr>
			<td>Nafion (un-mod.)</td>
			<td>19.93 ± 10.10</td>
		</tr>
		<tr>
			<td>TMTDA</td>
			<td>247.25 ± 12.49</td>
		</tr>
		<tr>
			<td>TBAB</td>
			<td>152.27 ± 5.29</td>
		</tr>
		<tr>
			<td>TMDDA</td>
			<td>262.05 ± 6.26</td>
		</tr>
		<tr>
			<td>TMOA</td>
			<td>129.18 ± 2.31</td>
		</tr>
		<tr>
			<td>TMDA</td>
			<td>141.23 ± 1.97</td>
		</tr>
		<tr>
			<td>TMHA</td>
			<td>131.75 ± 2.89</td>
		</tr>
		<tr>
			<td>TMHDA</td>
			<td>132.50 ± 1.18</td>
		</tr>
		<tr>
			<td>TMODA</td>
			<td>136.50 ± 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="Cell membrane diagram; quaternary ammonium addition showing protein-lipid interactions." src="/files/ftp_upload/3949/3949fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Schematic of TBAB incorporation into Nafion polymer and subsequent use in enzyme immobilization.
<img alt="Hexagonal petri dish with bacterial culture growth for static equilibrium analysis experiment." src="/files/ftp_upload/3949/3949fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
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.

<ol><li>Calabrese-Barton, S., Gallaway, J., Atanassov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzymatic+biofuel+cells+for+implantable+and+microscale+devices+Chem%5BTitle%5D)+AND+%22Rev%22%5BJournal%5D)">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/pubmed?term=((Enzymes+as+Working+or+Inspirational+Electrocatalysts+for+Fuel+Cells+and+Electrolysis%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzyme-Based+Biofuel+Cells%5BTitle%5D)+AND+%22Curr.+Opin.+Biotechnol%22%5BJournal%5D)">Enzyme-Based Biofuel Cells.</a> Curr. Opin. Biotechnol. 18, 228-234 (2007).</li>
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<li>Martinek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+of+the+Membrane+Environment+of+Enzymes%3A+Superactivity+of+Laccase+Entrapped+into+Surfactant+Reversed+Micelles+in+Organic+Solvents%5BTitle%5D)+AND+%22Biokhimiya%22%5BJournal%5D)">Modeling of the Membrane Environment of Enzymes: Superactivity of Laccase Entrapped into Surfactant Reversed Micelles in Organic Solvents.</a> Biokhimiya. 53, 1013-1016 (1988).</li>
<li>Moore, C. M., Akers, N. L., Hill, A. D., Johnson, Z. C., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+the+Environment+for+Immobilized+Dehydrogenase+Enzymes+by+Modifying+Nafion+with+Tetraalkylammonium+Bromides%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">Improving the Environment for Immobilized Dehydrogenase Enzymes by Modifying Nafion with Tetraalkylammonium Bromides.</a> Biomacromolecules. 5, 1241-1247 (2004).</li>
<li>Schrenk, M. J., Villigram, R. E., Torrence, N. J., Brancato, S. J., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Mixture+Casting+Nafion+with+Quaternary+Ammonium+Bromide+Salts+on+the+Ion-Exchange+Capacity+and+Mass+Transport+in+the+Membranes%5BTitle%5D)+AND+%22J.+Membr.+Sci%22%5BJournal%5D)">Effects of Mixture Casting Nafion with Quaternary Ammonium Bromide Salts on the Ion-Exchange Capacity and Mass Transport in the Membranes.</a> J. Membr. Sci. 205, 3-10 (2002).</li>
<li>Akers, N. L., Moore, C. M., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+Alcohol%2FO2+Biofuel+Cells+Using+Salt-Extracted+Tetrabutylammonium+Bromide%2FNafion+Membranes+to+Immobilize+Dehydrogenase+Enzymes%5BTitle%5D)+AND+%22Electrochim.+Acta%22%5BJournal%5D)">Development of Alcohol/O2 Biofuel Cells Using Salt-Extracted Tetrabutylammonium Bromide/Nafion Membranes to Immobilize Dehydrogenase Enzymes.</a> Electrochim. Acta. 50, 2521-2525 (2005).</li>
<li>Sokic-Lazic, D., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Citric+Acid+Cycle+Biomimic+on+a+Carbon+Electrode%5BTitle%5D)+AND+%22Biosens.+Bioelectron%22%5BJournal%5D)">Citric Acid Cycle Biomimic on a Carbon Electrode.</a> Biosens. Bioelectron. 24, 939-944 (2008).</li>
<li>Arechederra, R. L., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complete+Oxidation+of+Glycerol+in+an+Enzymatic+Biofuel+Cell%5BTitle%5D)+AND+%22Fuel+Cells%22%5BJournal%5D)">Complete Oxidation of Glycerol in an Enzymatic Biofuel Cell.</a> Fuel Cells. 9, 63-69 (2009).</li>
<li>Germain, M., Arechederra, R. L., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nitroaromatic+Actuation+of+Mitochondrial+Bioelectrocatalysis+for+Self-Powered+Explosive+Sensors%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Nitroaromatic Actuation of Mitochondrial Bioelectrocatalysis for Self-Powered Explosive Sensors.</a> J. Am. Chem. Soc. 130, 15272-15273 (2008).</li>
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<li>Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., Klenk, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+protein+using+bicinchoninic+acid%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150, 76-85 (1985).</li>
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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><tbody><tr><td>Nafion</td><td>Sigma-Aldrich</td><td>70160</td><td></td></tr><tr><td>Tetra alkylammonium bromide salts</td><td>Sigma-Aldrich</td><td>n/a</td><td></td></tr><tr><td>Alcohol dehydrogenase</td><td>Sigma-Aldrich</td><td>A3263</td><td></td></tr><tr><td>Nicotinamide adenine dinucleotide (NAD)</td><td>Simga-Aldrich</td><td>N7004</td><td></td></tr><tr><td>Sodium pyrophosphate</td><td>Sigma-Aldrich</td><td>P8010</td><td></td></tr><tr><td>Phenazine methosulfate (PMS)</td><td>Sigma-Aldrich</td><td>P9625</td><td></td></tr><tr><td>2,6-Dichloroindophenol (DCIP)</td><td>Sigma-Aldrich</td><td>D1878</td><td></td></tr><tr><td>Glucose oxidase</td><td>Sigma-Aldrich</td><td>G7141</td><td></td></tr><tr><td>4-Hydroxybenzoic acid</td><td>Sigma-Aldrich</td><td>240141</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>Peroxidase</td><td>Sigma-Aldrich</td><td>P8375</td><td></td></tr><tr><td>4-aminoantipyrine</td><td>Sigma-Aldrich</td><td>06800</td><td></td></tr><tr><td>UV/Vis Spectrophotometer</td><td>Thermo</td><td>Evolution 260 Bio or Spectronic Genesys 20</td><td></td></tr><tr><td>Vortex Genie</td><td></td><td></td><td></td></tr><tr><td>Analytical balance</td><td></td><td></td><td></td></tr></tbody></table>

hydrophobic salt-modified nafion for enzyme immobilization and stabilization

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.

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 ...

The overall goal of this procedure is to synthesize a my cellar, enzyme immobilization and stabilization polymer. To accomplish this first, combine the neon suspension with the cheal ammonium bromide salt, and vortex, the mixture in order to exchange the proteinated form of nafion with ammonium salts. The second step is to dry the resulting polymer by pouring the mixture into a whey boat and allowing the solvents to evaporate.Next, soak the polymer and deionized water for 12 to 24 hours to remove excess salt After salt extraction, rinse the polymer with water and then allow the polymer to dry completely by evaporation. The final step is to transfer the polymer to a clean vial and resuspend it insolvent for use in enzyme Im mobilization. Ultimately, this my cellar enzyme immobilization polymer is used to improve the stability of enzymes in activity assays.Generally, individuals new to this method will struggle because this procedure seems extremely straightforward, but the drying procedure is critical to the fabrication of a functional polymer. Demonstrating the procedure will be shu a graduate student in my laboratory To begin the procedure for modifying nafion with Nerium ammonium salts. Measure a threefold molar excess relative to the sul acid groups on the nafion polymer of the alk ammonium bromide salt.Transfer the alk ammonium bromide salt to a glass vial vigorously. Shake a bottle of 5%weight per volume, nafion suspension for approximately 30 seconds to ensure that the neon is suspended uniformly in solution. Pipette out two milliliters of the now resuspended nafion and add it to the glass vial containing the AGU ammonium bromide salt.Vortex the vial at 900 RPM for 10 to 15 minutes. Next, pour the viscous solution into a plastic weighing tray and use a pipette to transfer any residual solution from the vial to the weighing tray. In this example, the way boat is partially covered and left on the laboratory bench to allow the solvents to completely evaporate out of the weighing tray at a rate of evaporation that takes more than six hours.Complete evaporation of all solvent at the appropriate rate of evaporation should leave behind a yellow or brown transparent film on the bottom of the weighing tray as shown here. If the solvent evaporates too fast, a crusty or crystalline material will form instead of a transparent film indicating that the mic cellar structure of the polymer has been destroyed. If solvent evaporation is too slow or the ammonium salt is allowed to react too long and take on too much water vapor, either a tacky orange gel or a white crystalline material will form.In either case, the procedure must be restarted. After all solvent has evaporated at the appropriate rate of evaporation, fill the weighing tray with 10 to 20 milliliters of 18 mega ohm centimeter deionized water cover, and soak for 12 to 24 hours to remove excess alk ammonium bromide salts and HBR 12 to 24 hours later, pipette out the water and add enough deionized water to fill the tray. Be careful to always pipette away from the polymer film to avoid losing any of it.Rinse a total of three times after the rinses. Allow the weighing tray to sit uncovered until the polymer is completely dry. When completely dry, the polymer should be a clear and somewhat brittle plastic film.In contrast, polymers that were not dried properly will have a very different appearance after salt extraction as shown here. Using a spatula carefully remove the dried film from the weighing tray and transfer it into a clean glass vial. Add two milliliters of ethanol and three ceramic mixing beads and vortex for four hours or until the polymer film is completely resuspended.The alk ammonium modified nathe on solution is now ready to be used for enzyme immobilization. If the enzyme to be immobilized is a dry enzyme, weigh out one milligram of the enzyme into a 1.5 milliliter micro centrifuge tube and add one milliliter of a hundred millimolar pH seven phosphate buffer to create a one milligram per milliliter enzyme solution. Transfer 120 microliters of the one milligram per milliliter enzyme solution to a new tube.Add 60 microliters of the ALK ammonium modified nafion solution and vortex for 10 seconds. The enzyme to polymer solution ratio must be kept at two to one if this mixture needs to be scaled up for large numbers of replicates. Finally pipette 60 microliters of the enzyme polymer solution into the bottom of each of the three separate one square centimeter cuvettes and allow them to dry overnight.The stability of the enzyme within the polymer film can then be assessed by enzymatic activity assays as demonstrated. Next to begin this assay, add the following reagents into the vet co casted with the modified nafion polymer and NAD dependent dehydrogenase enzyme. 1.3 milliliters of 50 millimolar sodium pyrophosphate, 1.5 milliliters of freshly prepared 15 millimolar nicotinamide adenine dinucleotide or NAD and 0.1 milliliters of water.Place the vete in a UV visible spectrophotometer set to a wavelength of 340 nanometers zero. The spectrometer add 0.1 milliliters ethanol into the vete and mix reagents by gently pipetting the solution up and down five times for a blank use 0.1 milliliters of additional water. Instead of the 0.1 milliliters of ethanol, five minutes after the reagents were added to the vete record the absorbance at 340 nanometers.Record the absorbance again 20 minutes after adding reagents to the qve, these two data points will be plotted to get a slope that can be used for activity calculations to assay for enzymatic specific activity of immobilized pq. Dependent dehydrogenases pipette these reagents into the qve co casted with the modified nafion polymer and PQQ dependent dehydrogenase. 1.5 milliliters of sodium phosphate and 200 microliters of 600 micromolar pH methyls sulfate or PMS.Place the vete in a UV visible spectrophotometer set to a wavelength of 600 nanometers and zero. The spectrometer add 100 microliters of a mixture of 700 microliters to six chloro endemol or DCIP and 200 microliters of the substrate of interest. Mixed the reagents by gently pipetting the solution up and down five times for a blank.Use 200 microliters of water instead of the substrate of interest. Five minutes after the reagents were added. Record the absorbance at 600 nanometers.Record the absorbance again 20 minutes after adding the reagents to begin this assay. Add to the Q VET co casted with the modified nafion polymer and glucose oxidase. Two milliliters of a solution containing the following 0.2 molar para hydroxy benzoic acid 0.02%Sodium azide 128 units of peroxidase 0.3 millimolar, four amino anti pyre, one molar potassium phosphate, and 50 millimolars of glucose.Makes the solution by pipetting up and down five times. Place the Q vet in a UV visible spectrophotometer set to a wavelength of 510 nanometers. Five minutes after the reagents were added to the qve.Record the absorbance at 510 nanometers. Then record the absorbance again at 20 minutes after addition of reagents. This table shows assay results of NAD dependent glucose dehydrogenase activity immobilized and selected modified nafion polymers.It was observed that immobilized NAD dependent glucose dehydrogenase had lower specific activity than the enzyme and buffer solution. This is because polymer membranes decrease transport of large molecules, and although glucose the substrate for the assay is small, the co-enzyme NAD needs to diffuse in and out of the membrane. For NAD dependent dehydrogenase, the transport limitation due to immobilization in the polymer decreases the observed enzymatic activity.This next table shows assay results of PQQ dependent glucose dehydrogenase activity immobilized and selected modified nafion polymers. It's important to note here the higher activity of the enzymes immobilized on most nafion films versus the enzymes and buffer solution. Since PQQ dependent glucose dehydrogenase is a membrane associated protein, the hydrophobic modified my cellar, nafion provides a more membrane like environment than buffer for stabilizing the active PQQ dependent glucose dehydrogenase, thus enhancing the activity of the enzyme.Lastly, the representative assay results of glucose oxidase specific activity. Immobilized and selected modified nafion polymers are shown in this table. The enzymatic assays for each of the polymers with each of the three enzymes show that the trends in relative specific activity compared to enzyme and solution is a function of the enzyme system.Consequently, the exact polymer needed for each enzyme has to be optimized. While attempting this procedure, it is important to remember that the drying step is critical to this particular procedure.

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