Alok Singh and Walter J. Dressick
Center for Bio/Molecular Science & Engineering Naval Research Laboratory, Washington DC 20375

Lecture presented at Techtextil-Symposium North America 2006

I. Abstract

Thin catalytic films have been deposited on a variety of surfaces, including textiles, with a goal to develop self-cleaning surfaces. Films comprise a thin, layered, composite coating incorporating bioactive macromolecules in polyelectrolyte multilayers. Bioactive species in multilayers showed efficient, continuous toxin degradation. The pesticide hydrolyzing enzyme, organophophorous hydrolase (OPH), deposited on woven cotton or glass fiber cloths, showed sustained pesticide hydrolysis activity for at least twelve cycles over a period of three weeks. Coatings on cotton threads and fabric knitted from them performed similarly against the pesticide methyl parathion (MPT). The activity of enzymes-multilayers coatings was independent of temperature, pH and the presence of organic solvents (e.g.; methanol, acetone). Multilayers on β-cyclodextrin grafted cotton fabric showed a 2.5 times enhancement in catalytic activity for pesticide hydrolysis compared to the same multilayers deposited directly onto the cotton cloth. The simple multilayer deposition process and ability to incorporate multiple components in the layers makes the overall process attractive for commercial application.

II. Introduction

Pesticides are widely-used, hazardous chemicals outside the laboratory environment. Increased awareness by the populace concerning the hazards of pesticides and chemical pollutants, coupled with the growing threat of exposure due to terrorist action, long-term environmental accumulation, or accidental spills, requires the development of new countermeasures and remedial methods. Current protection gears (e.g.; clothing, filters) are not adequate as they rely on the use of physical barriers. Materials like activated charcoal and high efficiency particulate absorbing (HEPA) filters concentrate the hazardous materials and their decomposition by-products and require costly periodic replacement and disposal. Similarly, special clothing and gloves made from impermeable barriers have limited effectiveness due to their bulk/weight, cost, re-usability and safe disposal. Consequently, there is an urgent need to develop self-cleaning hazmat-free systems that address the concerns for handling and neutralizing such toxins in both water and air. Literature surveys reveal ongoing efforts focusing on the development of active protection systems involving reactive reagents and catalysts such as oxidants (titanium dioxide) [1] and molecularly-engineered catalysts (metal chelators) [2]. Enzymes are better catalysts than their synthetic counterparts by virtue of substrate specificity and high turnover numbers (e.g.; OPH Kcat = 12,000 s-1), but they lack stability and depend on precise pH, medium and temperature control to function efficiently. Sustaining enzyme activity for practical applications by immobilization on surfaces through covalent bonding [3] or physical entrapment [4] is an active area research. However, substantial loss of enzyme activity is reported during processing steps.

Recently, we have reported that enzyme activity can be maintained under relatively harsher conditions and over long periods of times by immobilizing enzymes in polyelectrolyte multilayer assemblies [5] and the multilayer assemblies can be further stabilized by encasing them in a polymer net [6]. In this report we present an overview of ongoing efforts on developing self-decontaminating textiles by depositing organophosphorous hydrolase (OPH) – polyelectrolyte multilayers on various textile substrates.

III. Method and Materials

III.1. Materials:  All chemicals and reagents used were technical grade and were used as received. OPH (EC 3.1.8.1) was received as freeze-dried powder from Aberdeen Proving Ground, MD. Structures of polyelectrolytes, buffers and methylparathion (MPT) pesticide and its p-nitrophenol (PNP) degradation product are shown in Figure 1.
 

III.2. Methods:  Textiles derived from glass fiber or cotton were used as received. For some experiments, cotton textile was modified by grafting β-cyclodextrin (CD) before depositing enzyme-polyelectrolyte multilayers. Grafting was carried out on 10 cm x 10 cm sized cotton pieces by placing them in a hot dimethyl formamide solution (100 °C) containing CD and hexyl diisocyanate (1:9, mole/mole) for a period of ~ 4 h. After the reaction, cloths were removed from the reaction solution and washed with water and dried in vacuum oven at 80 °C. Polycyclodextrin (PCD) particles were prepared in the same manner by treatment of the CD substrate alone with hexyl diisocyanate in DMF. In the latter case, solid particles were recovered by precipitation from hexanes, washed with methanol using ultrasound agitation, and sized by sieving the ground powder.
 
Substrates were thoroughly washed and dried with Milli-Q-ultrapure (18.2 MΩ∙cm) water before commencing layer deposition. Multilayers were deposited by sequentially dipping the substrates in various polyelectrolyte, enzyme, and wash solutions as summarized in Figure 2 [7].


Enzyme activity of enzyme-multilayer assemblies was monitored by placing substrates with catalytic coatings in 100 μM MPT solution in CHES pH 8.6 buffer containing 15% methanol. Release of PNP due to enzyme mediated hydrolysis of the MPT was monitored over a regular time intervals by using UV spectrophotometer. Control experiments using untreated cloths or cloths coated with multilayers not containing enzyme resulted in negligible hydrolysis of MPT (i.e.; < 2 mM).

IV. Results and Discussion

Our approach involves fabrication of self-decontaminating, multifunctional thin films (< 50 nm) as low cost, lightweight alternatives to protect the people and the environment from exposure to chemical and biological toxins. The concept of multilayer synthesis using a layer-by-layer approach is well established [8]. Multilayer films are readily prepared by sequential adsorption of oppositely charged polyelectrolytes, such as polyethylenimine (PEI) and polyacrylic acid (PAA), onto substrates like cloth or beads from water via dipcoating or spraycoating. 

Design of these multilayer coatings takes advantage of complementary catalysts, such as metal complexes and enzymes, capable of hydrolyzing pesticides or nerve agents on contact. Metal complexes provide long lasting protection, albeit at low activity; e.g., the stable Cu[H2NCH2CH2NH2]22+ complex only slowly hydrolyzes methyl parathion (MPT) pesticide (~0.026 s-1) [2]. In contrast, organophosphorous hydrolase (OPH) enzyme rapidly hydrolyzes MPT (~50 s-1), but loses activity quickly as it denatures after coming in contact with aqueous environment. We have addressed the key issue of enzyme stability via polyelectrolyte (PE) multilayer film encapsulation [6]. By adjusting the PE chemistries, we provide an environment that stabilizes enzymes against denaturation while preserving activity [6, 7].

Figure 3 shows a structural cross-section of a generalized film of this type. Occasional replacement of a PE layer by a layer of charged adsorbent (e.g., SiO2 nanoparticles), metal complex (e.g., CuII), or OPH enzyme during fabrication creates a film having a tunable sandwich structure. Toxins such as MPT entering the film first encounter the OPH layer, where most is rapidly hydrolyzed to p-nitrophenol (PNP) anion and acid. Underlying layers of metal complexes provide a backup to the enzyme. Finally, an innermost adsorbent nanoparticle layer prevents toxin by-products from reaching the protected target.



IV.1. Enzyme multilayer formation and determination of catalytic activity of films:  To examine the formation of enzyme-multilayers films on the surface of textiles, we first fabricated enzyme-multilayer films on fused silica slides to: (1) verify enzyme deposition during film fabrication by UV spectroscopy, and (2) demonstrate activity of the films for hydrolysis of MPT in solution. As depicted in Figure 4 (top spectrum) the presence of OPH in the multilayers deposited on the slides is evident from the OPH absorbance ~268 nm. Activity of the enzyme in the films was monitored by monitoring the MPT hydrolysis product, PNP, at 405 nm over time at different temperatures. Figure 2 (bottom spectrum) shows the presence of PNP in solution produced by the hydrolysis of MPT by one of the coated fused silica slides.

 

IV.2. Effect of humidity on film catalytic activity:  It is known that the enzymes denature at room temperature in the presence of an aqueous environment. From an applications viewpoint, it is important to evaluate the activity of enzyme-multilayer coatings in environments having different humidity levels over a longer time period. Evaluations were carried out by placing glass beads (~ 50 μm diameter) bearing 5-OPH layer PE films in chambers with fixed and controlled levels of humidity. Standard, saturated inorganic salt solutions were used for maintaining controlled humidityenvironments. All experiments were carried out at room temperature. Figure 5 shows good initial catalytic activity over 15-85% humidity range.  Fractional loss of OPH activity was observed over the course of time and with increases in humidity. Interestingly, at 0% humidity some activity was observed; this was perhaps due to the presence of tightly bound water within polyelectrolyte multilayers.

IV.3. Development of poly β-cyclodextrin (PCD) beads for enzyme-multilayer coatings:  As mentioned earlier in the Introduction, our goal is to develop lightweight, efficient catalytic coatings that can not only hydrolyze the toxins but also absorb their hydrolysis products. PCD particles with the general structure shown in Figure 6 were synthesized with this goal in mind. PCD particles are four times lighter than silica particles, inert to acidic and basic media, and insoluble in most organic solvents. These properties make them suitable candidates for depositing enzyme multilayer films. These particles have cavity volume of 262 Ǻ3, 0.06 cm3/g pore volume and a surface area of 10.2 m2/g. Before conducting any film deposition experiments, particles were sonicated in methanol for 10 minutes to clean the pores. In a static competition experiment challenging PCD beads (particles) with MPT and PNP, MPT bound to the beads ~2.2 times better than PNP,
           
R = KM/KP = [M.PCD].[P]/{[P-PCD].[M]}= (43 μM).(74 μM)/{(57 μM) ∙ (26 μM)} ≡ 2.2

where R is the binding ratio, M is methylparathion and P is p-nitrophenol. PCD particles were used for depositing enzyme-multilayer films in the following sequence per Figure 2: PCD/(PEI-w/OPH-b/BPEI-b/PSS-w)4PEI-w/OPH-b/BPEI-b/PAA-w/EDA (where the suffixes “w” and “b” refer to materials dissolved in water or aqueous BTP pH 8.6 buffer solution, respectively). The catalytic activity of these beads was examined under different pH and solvent conditions. Our results are summarized in Table 1. Our results clearly indicate that PCD helped in improving the low temperature activity of the coatings, presumably by binding and concentrating MPT near the enzyme sites to partially offset the reduced enzyme activity at lower temperature, an important step towards making active coatings effective over a wider temperature range. Moreover, tolerance against stress agents and sustained activity in plain water further demonstrated the practical utility of our system.

IV.4. Self Cleaning Textile:  Cloths derived from woven fiber glass and cotton were coated with enzyme-multilayer polyelectrolyte films with and without surface modification and tested for their catalytic activity following the similar protocol used for silica and PCD beads. Cotton cloths were also modified with CD to achieve improved catalytic activity as observed in the case of PCD particles.

            IV.4.a. Self cleaning fiberglass cloths:  Following the protocol presented in Figure 2, catalytic cloths from glass fibers were prepared. Enzyme assays [6] of the coated glass cloth revealed deposition of 119 ng OPH/g cloth in 4 layer enzyme deposition. These cloths were brought in contact with 100 mL of 100 μM MPT for 22 hours and the formation of PNP was measured to determine the catalytic activity. For each cycle a fresh solution of MPT was used in the experiment. Figure 7 provides visual illustration of the hydrolytic activity of catalytic cloth after 3rd catalytic cycle was performed.

Table 1:  Summary of Catalytic Activities of Enzyme-Multilayer Glass Beads Under Different Conditions

Environment  (Solvent)	Initial Catalytic Velocity (V0) (x 10-8 L∙ mole-1∙ s-1)
80:20 CHES (aq.):MeOH (23 °C) 80:20 CHES (aq.):MeOH (4 °C) Water (pH 6.6; 23 °C) Tap water (23 °C) Methanol wash (2h) Acetone (2h) Salt stress 2M NaCl (2h)	19±2 14±2 36±4 11±1 0.6±0.1 1.8±0.2 1.0±0.2

            Figure 8 shows the reusability profile of glass cloth for 12 cycles over a period of three weeks. A temporary spike in film activity was observed following low temperature storage (4 °C) for the cloth containing a single OPH layer over the weekend. This spike was, however, essentially absent when the number of OPH layers increased from one layer to four layers. Preliminary experiments suggest that such behavior is related to the ability of less constrained polyelectrolyte layers in the single OPH layer film to adjust conformation with changing temperature, thereby affecting the conformation and activity of the trapped OPH [7]. Work is in progress to determine the mechanism of this behavior.

            IV.4.b. Self cleaning cotton cloths:  Samples of cotton cloths were coated with a spray technique in addition to the dipcoating technique applied in all previous experiments. Spraycoating is a viable alternative fabrication mode, which manufacturers may find attractive. However, results demonstrated that dipcoated cloth is 6-7 times more active than spraycoated cloth (see Figure 9 for a visual comparison). The term “7-cycles” in the figure indicates seven cycles without changing the MPT solution; this was done to make comparison realistic. This difference is due, at least in part, to: (1) lower contact times for deposition solutions during spraycoating (i.e.; 2 min) compared to dipcoating (i.e.; 10 min), and (2) possible depletion of reagent in the thin liquid film contacting the substrate during spraycoating. It is also interesting to note that OPH coated cotton cloth is ≥2 times more active than equivalent fiberglass cloth. This likely reflects differences in surface hydroxyl concentrations available for interaction with the film components between the two substrates and/or the porous nature of the cotton fiber.  Additional work is in progress to further understand these loading differences.




























































IV.4.c. Self cleaning cotton cloths decorated with β-cyclodextrins:  We have shown in section IV.3 that PCD particles are lighter in weight and show improved catalytic activity over silica particles. Because cotton’s molecular structure also contains reactive hydroxyl groups, CD can also be directly grafted to its surface via our diisocyanate chemistry. Our goals in modification of the cotton surface in this manner are: (1) to improve the catalytic efficiency of the cloths, and (2) remove the MPT hydrolysis products by adsorbing them in CD cavities.  Following the activity measurement protocols described earlier, we compared the catalytic activities of plain cotton cloth, cotton cloth coated with a PE film bearing 4-OPH layers and the CD grafted cotton cloth coated with a PE film bearing 4-OPH layers. Figure 10 shows the results for these three pieces. While the control uncoated cloth did not show any activity, the cotton cloth coated with a PE film containing OPH showed excellent activity by hydrolyzing ~40% of the MPT within ~ 1 day. In contrast, as expected based on our previous experiments using PCD particles, the catalytic efficiency of the CD-grafted cotton cloth was increased by a factor of ~2.5, with essentially complete hydrolysis of the MPT occurring within ~ 1 day in solution.

V. Summary and Conclusions

Reproducible enzyme multilayer film fabrication methods have been established. Proper selection and nature of the polyelectrolyte “spacer” layers before and after enzymes maximize OPH (EC 3.1.8.1) loading in the films [7]. Multilayer films can be easily deposited on a variety of substrates (e.g.; glass, cotton, poly- b-cyclodextrin) by multiple deposition methods, such as dipcoating, spraycoating, spincoating (not discussed here), depending on the need and nature of the application. Results have clearly demonstrated that enzyme encapsulated in multilayers in this manner is sufficiently stable and active under broad humidity and storage conditions for use as a toxin decontaminant. Catalytic activity increases monotonically with number of enzyme layers. In addition, enzyme denaturation in harsh user environments (e.g.; presence of organic solvents or aqueous salt solution) can be further reduced by stabilization of multilayers via capping of the outermost layer. Finally, we have demonstrated enzyme performance, stability, and re-usability (at least 3 weeks; 12 re-use cycles) under various practical conditions, including pure aqueous systems, as well as at low temperature using b-cyclodextrin modified cloth substrates. Work is currently in progress for developing complete protection system against chemical and biological toxins coupled with internal end-of-active life indicators.

VI. Acknowledgement

This research was funded by the Office of Naval Research through NRL core 6.2 research funds.

Contact information:  Corresponding author: Dr. Alok Singh, Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue, SW Washington DC 20375.  Phone: 202-404-6060, email: alok.singh@nrl.navy.mil.

VII. References

1. W.A. Daoud, J.H. Xin, Y.H. Zhang, Surf. Sci. 2005, 599, 69.
2. C. Hartshorn, A. Singh, E. L. Chang, J. Mater. Chem., 2002, 12, 602.
3. K. Ogawa, B. Wang, E. Kofukuta, Langmuir, 2001, 31, 1664.
4. a) F. Caruso, D. Trau, H. Moehwald, R. Rennenberg, Langmuir, 2000, 16, 1485; b) Y. Lvov,
K. Ariga, T. Kunitake, Chem. Lett., 1994, 2223; c) J. D. Hong, K. Lowack, J. Schmidt, G. Decher, Prog Colloid Polym. Sci., 1993, 93, 98.
5. J. P. Santos, E. R. Welsh, B. P. Gaber, A. Singh, Langmuir, 2001, 17, 5361.
6. Y. Lee, I. Stanish, V. Rastogi, T. C. Cheng, A. Singh, Langmuir, 2003, 19, 1330.
7. A. Singh, Y. Lee and W. J. Dressick, Adv. Mater. 2004, 16, 2112.

8. G. Decher, Science 1997, 277, 1232.