Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Summary

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.

Abstract

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.

Protocol

1. Modification of Nafion with Quaternary Ammonium Salts

1. Shake a bottle of 5% w/v Nafion suspension vigorously for approx. 30 seconds to ensure that Nafion is suspended uniformly in solution.
2. 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).
3. 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.
4. Vortex the vial at 1500 rpm for 10-15 minutes.
5. Pour the viscous solution in....

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nafion	Sigma-Aldrich	70160
Tetra alkylammonium bromide salts	Sigma-Aldrich	n/a
Alcohol dehydrogenase	Sigma-Aldrich	A3263
Nicotinamide adenine dinucleotide (NAD)	Simga-Aldrich	N7004
Sodium pyrophosphate	Sigma-Aldrich	P8010
Phenazine methosulfate (PMS)	Sigma-Aldrich	P9625
2,6-Dichloroindophenol (DCIP)	Sigma-Aldrich	D1878
Glucose oxidase	Sigma-Aldrich	G7141
4-Hydroxybenzoic acid	Sigma-Aldrich	240141
Sodium azide	Sigma-Aldrich	S8032
Peroxidase	Sigma-Aldrich	P8375
4-aminoantipyrine	Sigma-Aldrich	06800
UV/Vis Spectrophotometer	Thermo	Evolution 260 Bio or Spectronic Genesys 20
Vortex Genie
Analytical balance