A New Surface for Immobilizing and Maintaining the Function of

Applied and Plasma Physics, School of Physics (A28), The University of Sydney, Sydney, NSW 2006, Australia. Biomacromolecules , 0, (), ... ACS Members...
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Biomacromolecules 2009, 10, 2577–2583

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A New Surface for Immobilizing and Maintaining the Function of Enzymes in a Freeze-Dried State Neil J. Nosworthy,* David R. McKenzie, and Marcela M. Bilek Applied and Plasma Physics, School of Physics (A28), The University of Sydney, Sydney, NSW 2006, Australia Received May 7, 2009; Revised Manuscript Received June 26, 2009

We describe a new surface produced by plasma treatment for immobilizing proteins in the dry state. The need for surfaces suitable for immobilizing proteins is increasing because of demand for microarray diagnostic services, biosensors, and chemical processing. Storage of surface attached proteins in the dry state offers benefits of long shelf life, protection from proteases, easier transportation and convenient storage. In this work, we produced plasma-modified polyethylene surfaces and tested them using two important enzymes for which convenient functional assays are available, namely, horseradish peroxidase and catalase. Over 80% of the function of horseradish peroxidase is retained after freeze-drying, and this function is unaltered after 4 months of storage at 4 °C on the treated polyethylene surface. The factors important for maintenance of surface attached enzyme stability were (1) plasma immersion ion implantation (PIII) treatment of the surface, (2) freeze-drying with sucrose in the buffer solution, (3) dry storage with desiccant, and (4) maintaining the freeze-dried protein at a reduced temperature. Other than sucrose, no other additives are needed.

1. Introduction The attachment of proteins to surfaces is used in diagnostic assays such as ELISA, protein arrays, drug delivery systems, antibody arrays, motility assays, and biosensors.1-3 The utility of diagnostic tests and biosensors is increased by the ability to store the surface-attached proteins for long periods while maintaining the biological activity of the protein until required.4,5 In solution, proteases reduce the lifetime of proteins and reactions such as oxidation can take place, initiating unfolding. A protein immobilized on a surface may inherently have a lower stability than the same protein in solution and so may be more vulnerable to a loss of function. Therefore, storage in solution of surfaces with attached protein will limit their useful life. Dry storage, if it can be achieved, would be beneficial for increasing the useful life of protein-based products and devices. In addition, transportation and storage of dried protein is more convenient and cost-effective than transportation in the hydrated state. Freeze-drying has been used as a method of dry storage for drugs, foods, and proteins and, for some proteins, enables almost permanent storage.6 Transport of proteins in the dry state is easier as there is less chance of the protein being denatured by rough handling. The ability to freeze-dry proteins and maintain their function on a solid surface would be of significant economic value. Antibodies tend to denature on hydrophobic polystyrene surfaces,7 and simple air-drying of the surfaceattached antibody enhances the denaturing effect.8 If the antibody could be stabilized by freeze-drying, this would extend the shelf life of antibody-based products such as diagnostic arrays. In plasma immersion ion implantation (PIII) of polymer surfaces, a negative voltage is applied to a target material immersed in a glow discharge plasma, accelerating ions from the surrounding plasma and causing them to be implanted * Corresponding author. Address: Dept. Anatomy and Histology, Anderson Stuart Bldg F13, University of Sydney, Sydney, NSW, 2006, Australia. Ph: 61-02-93516543; fax: 61-02-93516546; e-mail: neiln@ anatomy.usyd.edu.au.

beneath the surface.9 PIII treatment of polymers changes their surface structure and properties. It creates free radicals, which react with each other to form a highly cross-linked subsurface and with oxygen in the atmosphere to produce a higher energy, more wettable surface.10 The treatment is simple, and any polymeric surface can be modified by PIII treatment. Proteins have been shown to bind covalently to PIII-treated polyethylene (PE) and polystyrene surfaces using a simple incubation step without the need for linker molecules and to maintain their biological function over extended periods of time of storage in fresh buffer.11-15 While we have demonstrated that surfaceattached protein can be maintained in a functional state in the hydrated condition for periods of the order of several weeks, this time may not be long enough for shelf storage of highvalue added products that use surface-attached protein, such as biosensors or diagnostic arrays. In this paper we investigate the hypothesis that PIII treatment of polymer surfaces may enhance the stability of proteins immobilized on them in the dry state. We use PE as an example polymer and seek to identify optimum parameters for the freeze-drying process. There have been reports of oven drying, air-drying, spin-drying, vacuum drying, critical point drying, or freeze-drying of proteins on surfaces4,5,16-22 as ways of maintaining the stability of proteins in the dry state. However, to our knowledge, there have been no studies of the stability of proteins freeze-dried on plasma-treated polymer surfaces.

2. Experimental Section An ultrahigh molecular weight (UHMW) PE sheet (200 µM thick) was purchased from Goodfellow Cambridge Ltd. (cat. no. ET301200/ 1) and cut into rectangles of dimensions 13 mm × 15 mm. Horseradish peroxidase (HRP; catalogue number P6782), 3,3′,5,5′-tetramethylbenzidine (TMB; T0440) and catalase (C3155) were from Sigma. All other reagents were of analytical grade. Nitrogen was 99.99% pure. 2.1. Plasma Treatment of PE. An inductively coupled radio frequency (RF) plasma treatment system operated at 2 mTorr nitrogen (99.99%) was used to modify the polymer surfaces. The system is

10.1021/bm900523m CCC: $40.75  2009 American Chemical Society Published on Web 07/30/2009

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described in detail elsewhere.12,14 The surface modification methods used in this work are identical to those used previously that have been shown to produce covalent attachment of proteins. The metal sample holder was 10 cm in diameter. Two kinds of nitrogen plasma treatment were used. In the first treatment, PE was exposed to the RF plasma with no electrical connections made to the sample holder. In other words, the sample holder was allowed to find its own “floating” potential. The second treatment process was a PIII process and was used for the majority of experiments. Pulses (20 kV) of duration 20 µs at a repetition rate of 50 Hz were applied to a cylindrical mesh mounted over the sample at a distance of 30 mm to accelerate ions for implantation into the surface. The mesh was grounded between pulses. PIII treatment differs from conventional plasma treatment by implanting ions with energies of tens of kiloelectron volts into the polymer surface, resulting in a modification of the polymer to greater depths. The sample was exposed to the RF plasma as in the first process during the pulse off time. The total treatment time for both types of plasma treatment was 800 s unless otherwise stated. 2.2. Attachment of HRP to Plasma-Treated and Untreated UHMW PE. HRP was diluted to 50 µg/mL in 10 mM sodium phosphate buffer pH 7 (PO4 buffer). The PE samples were incubated in the protein solution overnight with rocking in 75 mm sterile Petri dishes with the samples floating on the surface with the treated side face down. After incubation, HRP was removed from the Petri dish, and 15 mL of wash buffer (PO4 buffer) was added and left for 20 min. Samples were then transferred to 50 mL Falcon tubes containing 40 mL o fresh PO4 buffer and rocked for 20 min. This was repeated four times. For samples in which sucrose was used, an additional wash was carried out in PO4 buffer containing various concentrations of sucrose. The samples were then freeze-dried. 2.3. Freeze-Drying of HRP-Coated UHMW PE. Two ways of freezing the protein on the PE surface were compared in initial tests. In the first method, we immersed the HRP-coated PE surfaces directly in liquid nitrogen in a Falcon tube. When most of the liquid nitrogen had boiled away, the Falcon tube was transferred immediately to vacuum in the freeze-drier. In the second method, samples in 50 mL Falcon tubes containing 15 mL of PO4 buffer with or without sucrose were placed into liquid nitrogen, and the samples were frozen while swirling the Falcon tube. The swirling motion tended to suck the samples into the PO4 buffer, ensuring the samples were trapped completely within the frozen ice. Once frozen, the samples were transferred to a Dynavac FD1 freeze-drying chamber and a vacuum applied for 48 h. Unless otherwise stated, the second method was used. The freeze-dryer had no temperature control, so secondary drying was carried out at room temperature (23 °C). The samples were removed from the Falcon tube and placed into a 15 mL plastic tube, and this was placed inside a 50 mL Falcon tube with or without desiccant and sealed. For samples that were desiccated, freshly activated silica gel was added until the volume reached the 10 mL indicator line on the Falcon tube. Samples stored at a nominal 4 °C were placed inside a 4 °C cold room and samples stored at a nominal -20 °C were placed in a -20 °C freezer. Room-temperature samples were stored at 23° ( 2 °C. 2.4. HRP Functional Assay. To determine the functionality of the rehydrated enzyme after various storage periods in the freeze-dried state, a colorimetric assay was used. The freeze-dried samples were placed in PO4 buffer for 10 min to rehydrate before testing for HRP activity. The protein-coated PE samples were then clamped between two stainless steel plates separated by an O-ring (inner diameter 8 mm, outer diameter 11 mm), which sealed to the plasma-treated surface, and a 7 mm hole was in one of the plates. The HRP substrate, TMB (75 µL), was added through the hole, and the blue color was allowed to develop. After 30 s, 25 µL of oxidized TMB was removed and added to 50 µL of 2 M HCl. The addition of acid yields a yellow end product. An additional 25 µL of unreacted TMB was then added, bringing the total volume to 100 µL. The absorbance at 450 nm was measured using

Figure 1. Activity of HRP on the surface of PIII-treated and untreated PE and stored in solution at temperatures of at 4 and 23 °C for 0, 3, 10, 30, and 60 days. (filled) PIII treated PE; (open) untreated PE; (upward striped) 23 °C; (downward striped) 4 °C.

a spectrophotometer (Beckman Lifescience DU530). The HRP activity test was carried out at room temperature (23° ( 2 °C). 2.5. Catalase Functional Assay. Catalase was attached to PE and freeze-dried using the same protein concentration and buffer conditions as for HRP in PO4 buffer containing 2.5% sucrose. After freeze-drying, catalase was rehydrated in PO4 buffer for 10 min, and surfaceactive catalase was assayed as previously described.14 Briefly, PEattached catalase was assayed by clamping the samples between two stainless steel plates separated by an 0-ring as for the HRP assay. Catalase activity was assayed by adding 75 µL of 6 mM hydrogen peroxide in PO4 buffer and allowed to incubate for 6 min. Remaining hydrogen peroxide in PO4 buffer was assayed at 475 nm by removing 3 µL and assaying the amount left by a modification of Cohen’s method.23 Data was plotted by subtracting the absorbance remaining from the absorbance that was obtained from assaying the initial 6 mM H2O2 assay solution. Plotted this way, the results are comparable to the HRP figures. That is, higher absorbance values show more active protein. The activity test was carried out at room temperature (23° ( 2 °C).

3. Results 3.1. Storage of Surface-Attached Protein in Solution. In order to provide a baseline for the activity of enzyme stored in solution after surface attachment, we measured the activity using a colorimetric assay after various storage times. Figure 1 shows the activity of surface-attached HRP as a function of time stored in PO4 solution. After HRP attachment and washing in PO4 buffer, the surfaces were stored at two temperatures: 4 and 23 °C. The PIII-treated surfaces maintained 78% of their initial activity after 60 days when the solution was kept at 4 °C. The untreated surface maintained 50% of the initial activity under the same conditions. For a storage temperature of 23 °C, the activity was almost zero for all surfaces after 60 days, with the untreated surface showing the most rapid decline. 3.2. Activity after Air-Drying. After HRP attachment and washing, the samples were removed from the phosphate buffer and left on the bench at room temperature for 4 h to dry. Figure 2 shows the activity measured after the dry surfaces are rehydrated in buffer solution. The enzyme function is substantially less than the activity of the same surface-attached enzyme stored in solution. If 2.5% sucrose is included in the final rinse

New Surface for Immobilizing Proteins in the Dry State

Figure 2. Activity after air-drying. (A) PIII-treated PE without drying. (B) Untreated PE without drying. (C) PIII-treated PE and attached HRP dried for 4 h on the bench. (D) Untreated PE and attached HRP dried for 4 h on the bench. (E) Same as C, but dried with added 2.5% sucrose. (F) Same as D, but dried with added 2.5% sucrose. (9) PIII treated PE; (0) untreated PE.

Figure 3. The activity after rehydration of surface-attached HRP freeze-dried in phosphate buffer with two different freezing methods: (1) snap frozen directly in liquid nitrogen and (2) samples were frozen while immersed in PO4 buffer. (A) PIII-treated PE before freeze-drying. (B) Untreated PE before freeze-drying. (C) PIII-treated PE with HRP snap frozen directly in liquid nitrogen. (D) Untreated PE with HRP snap frozen directly in liquid nitrogen. (E) PIII-treated PE with HRP frozen in phosphate buffer. (F) Untreated PE with HRP frozen in phosphate buffer.

solution prior to air-drying, a marginal improvement of HRP activity was observed on the PIII-treated surfaces but not on the untreated surface. Even after this improvement, the activity of the enzyme on the PIII surface is less than that on the untreated surface, in contrast to the situation for the surfaces stored in solution. If samples were allowed to dry for 24 h, very little HRP activity could be detected on either the PIII or untreated PE surfaces (data not shown). 3.3. Activity after Freeze-Drying. Figure 3 compares the activity of the surface attached protein after freeze-drying using the two different freezing methods in phosphate buffer and rehydration. The two freezing methods gave similar results for both PIII-treated and untreated surfaces, within error limits. Freeze-dried samples retained approximately 50% of the activity

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Figure 4. The recovery of the activity of HRP, immobilized on PE after freeze-drying in solutions containing sucrose. Activity was measured immediately after rehydration. (0) HRP activity on PIIItreated PE before freeze-drying (BFD). (O) HRP activity on untreated PE before freeze-drying (BFD). (9) HRP activity on PIII-treated PE freeze-dried in the presence of sucrose (concentrations 0-2.5%). (b) HRP activity on untreated PE freeze-dried in the presence of sucrose (concentrations 0-2.5%).

of samples that were tested immediately after incubation without freeze-drying. Freeze-dried samples that were stored in laboratory air overnight showed little activity remaining (data not shown). Because it was thought that direct contact with liquid nitrogen could lead to contamination of the surface, freezedrying in buffer solution was adopted as the preferred method for all subsequent experiments. 3.4. Effect of Sucrose on Recovery of HRP Activity after Freeze-Drying. Figure 4 shows the activity on rehydration of samples immediately after freeze-drying with sucrose in the phosphate buffer as a function of the concentration of sucrose. There is a marked improvement in the recovery of HRP activity after rehydration when sucrose is present. For PIII-treated surfaces, recovery of HRP activity after freeze-drying improved from 55% to 80% at 0.25% sucrose. Increasing the sucrose concentration above 0.25% did not result in any further improvement in the recovery of HRP activity on PIII treated surfaces. For untreated surfaces, there was a gradual improvement from 45% to 60% recovery of initial activity with increasing sucrose concentration up to 2.5%. We used 2.5% sucrose for the subsequent experiments where sucrose was used. 3.5. Effect of Desiccation on Recovery of HRP Activity after Freeze-Drying. Figure 5 compares the activity of freezedried HRP on surfaces as a function of storage time when the storage takes place in a sealed Falcon tube with and without desiccant. The samples stored with desiccant showed a markedly superior recoverable HRP activity when rehydrated. The surfaces were freeze-dried with sucrose. In the case of PIII-treated surfaces, approximately 60% of the initial activity can be recovered after 10 days storage, whereas, for untreated surfaces, only 15% of the initial activity can be recovered. In a set of experiments with surfaces incubated without sucrose in the solution, activity was rapidly lost, whether desiccant was used or not (Figure 6). The untreated surface actually performed better than the treated surface in this case. There is a synergistic effect of PIII treatment with the presence of sucrose and storage in the presence of desiccant.

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Figure 5. Effect of desiccation on the recovery of HRP activity after freeze-drying with 2.5% sucrose for 3, 6, and 10 days and stored at 23 °C. BFD ) before freeze-drying; AFD ) after freeze-drying and immediately rehydrated and assayed. (filled) PIII treated; (open) untreated PE; (downward striped) plus desiccation; (upward striped) minus desiccation.

Figure 6. Effect of desiccation on recovery of HRP activity after freezedrying without sucrose for 1, 2, and 3 days at 23 °C. BFD ) before freeze-drying; AFD ) after freeze-drying and immediately rehydrated and assayed. (filled) PIII-treated PE; (open) untreated PE; (downward striped) plus desiccation; (upward striped) minus desiccation.

3.6. Effect of Plasma Treatment Conditions on the Recovery of HRP Activity after Freeze-Drying. We compared the effect of conventional plasma treatment with PIII treatment of PE. Figure 7 shows the recoverable activity of HRP on PIIItreated surfaces and those treated by conventional plasma methods. Sucrose was used in the buffer and desiccant was included in the storage vessel. PIII-treated surfaces are markedly superior to conventional plasma treated surfaces. After storage on PIII surfaces approximately 55% of activity remaining after 10 days compared to only 20% for conventional plasma treatment and 10% for untreated surfaces. After 30 days, 30% of HRP activity was recovered on PIII-treated surfaces, whereas activity was close to zero for both conventional plasma treated and untreated PE surfaces. PE was then PIII treated for different periods of time and tested for the amount of recoverable HRP activity after freezedrying. Figure 8 shows that longer PIII treatment times gave a better recovery of HRP activity when stored for 30 days than shorter treatment times. The immobilization yield of active

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Figure 7. Comparison of plasma treatment with or without PIII ion bombardment on the recovery of HRP activity after freeze-drying with 2.5% sucrose and stored desiccated for 3, 10, and 30 days at 23 °C. BFD ) before freeze-drying; AFD ) after freeze-drying and immediately rehydrated and assayed. (open) Untreated PE; (striped) plasma-treated PE; (filled) PIII-treated PE.

Figure 8. Effect of PIII treatment time on recovery of HRP freezedried with 2.5% sucrose and stored desiccated for the indicated number of days on PE at 23 °C. (open) 0 s (untreated PE); (upward striped) 20 s; (horizontally striped) 100 s; (downward striped) 300 s; (filled) 800 s.

protein is not affected by the treatment time since we see no change with treatment time in the activity measured for day 0 at each treatment time. We have also studied the functionality of HRP on PIII-treated PE as a function of storage time in solution, and we found that a PIII treatment time of around 400-800 s was optimum (Nosworthy, unpublished work) for long-term storage in solution. In the case of freeze-drying, the longest treatment time (800 s) was the best, and this differed slightly from the optimum time required for stability in solution; this was the treatment time used for all other figures in this paper. A potential problem for some applications that use optical detection through the polymer surface is the browning of the treated surface due to carbonization, significant at the longest treatment times.

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in error bars, while at 4 and 23 °C, 50% of the activity remains. For the untreated surfaces protein activity was low after 120 days.

4. Discussion

Figure 9. Effect of storage temperature on the recovery of HRP activity after freeze-drying with 2.5% sucrose and stored desiccated at three different temperatures. BFD ) before freeze-drying; AFD ) after freeze-drying and immediately rehydrated and assayed. (filled) PIII-treated PE; (open) untreated PE; (downward striped) 23 °C; (upward striped) 4 °C; (horizontally striped) -20 °C. (A) 3 days; (B) 10 days; (C) 30 days; (D) 60 days; (E) 120 days.

Figure 10. Effect of storage temperature on the recovery of catalase activity after freeze-drying with 2.5% sucrose and stored desiccated at three different temperatures. BFD ) before freeze-drying; AFD ) after freeze-drying and immediately rehydrated and assayed. (filled) PIII-treated PE; (open) untreated PE; (downward striped) 23 °C; (upward striped) 4 °C; (horizontally striped) -20 °C. (A) 3 days; (B) 10 days; (C) 30 days; (D) 60 days.

3.7. Effect of Cold Storage on Recovery of HRP Activity after Freeze-Drying. Figure 9 shows that storage at 4 or -20 °C gives a further improvement in the storage life of freeze-dried HRP on PIII-treated PE. After 4 months, there was no decrease in the activity of freeze-dried HRP if stored at -20 °C and the decrease in activity at 4 °C was very small with a recoverable activity falling only just outside the error bars of the control sample immediately after freeze-drying (AFD in the figure). 3.8. Recovery of Catalase Activity after Freeze-Drying on PE. Figure 10 shows the recoverable activity of catalase on PIII-treated surfaces stored with sucrose and desiccation. After 120 days at -20 °C, there is no detectable loss of activity with

We have found four conditions that work toward maintaining the function of HRP after it has been freeze-dried on a PE surface. The conditions generally, but not always, work additively in the sense that the benefit given by each one is maintained when an additional one is used. The conditions are as follows: 1. PIII treatment of the surface 2. Freeze-drying with sucrose in the buffer solution 3. Storage dry with desiccant after freeze-drying 4. Maintaining the freeze-dried protein at a reduced temperature. PIII treatment of a polymer surface used for attachment of protein has previously been observed to maintain the function of the protein better than an untreated surface when the surfaces are stored in buffer solution with periodic refreshing of the buffer.11,12 This beneficial effect of the PIII treatment has been demonstrated for the attachment of catalase,14 soybean peroxidase,13 HRP,12,15 and tropoelastin (Bax, D. V. Acta Biomater., in press). Covalent binding of the protein to the PIII-treated surface was confirmed by the resistance of the attached protein to removal by sodium dodecyl sulfate (SDS) detergent.13,14 Our current understanding of the mechanism of this covalent binding is also discussed in these publications. The results of this study show that PIII-treated surfaces allow better recovery of protein activity after storage in the freezedried state than untreated surfaces or surfaces treated by conventional plasma treatment without PIII (Figure 7). This advantage is maintained with or without storage in desiccant (Figure 5), with or without storage at reduced temperature (Figure 9) in the presence of sucrose but not without (Figures 5 and 6). The advantage over untreated surfaces and surfaces exposed to conventional plasma treatment is improved by increasing the fluence (proportional to the treatment time) of the PIII treatment (Figure 8). The fluence is a measure of the number of energetic ions impacting on the surface and scales linearly with treatment time. The best results were observed for the highest treatment time used in our experiment (800 s). The improved performance of PIII surfaces relative to conventional plasma-treated surfaces in maintaining the function of dry protein may be related in some way to their reduced hydrophobic recovery.24 PIII surfaces maintain their hydrophilicity better than plasma treated surfaces because the deeper treatment of PIII slows down hydrophobic recovery.24 Sucrose acts to maintain the protein function after rehydration on all surfaces (Figures 4, 5, and 6). There was a sharp increase in the recovery of HRP activity when sucrose was added on PIII -treated surfaces, but only a gradual improvement on untreated surfaces. Perhaps the presence of sucrose on the treated surfaces allows the protein to be more rigid as a result of better linking between sucrose, HRP, and the introduced oxygen molecules that were on the surface as a result of PIII treatment. The action of sucrose in maintaining the function of proteins conventionally freeze-dried in solution (rather than attached to a surface) has been widely reported. The mechanism is under current investigation, but hydrogen bonding between sugar and protein may be responsible for inhibition of dehydration-induced protein unfolding.25 The sugar may replace the hydrogen bonding between the protein and its bound water, facilitating the removal of the bound water during the freeze-drying step.

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The bound water if still present after freeze-drying may act as a plasticizer, allowing motion of the protein molecules and facilitating conformational change with consequent denaturation. The effect of removing bound water is therefore to raise the glass transition temperature, which is desirable for long-term storage.26 Note that there may an optimum concentration of sucrose as it has been found that increasing concentrations of sucrose can lead to a reduction in the glass transition temperature Tg of dried protein in solution.27,28 The preservation of tertiary structure during the drying process by the sucrose also appears to be important.28,29 More recently it has been suggested that fast local dynamics is the best predictor of stability.30 Storage of freeze-dried HRP on PE in a dry environment with a desiccant is always better than storage in laboratory air (Figures 5 and 6). When the protein solution is frozen, the protein-bound water remains separate from the ice phase.31 The removal of bound water from the protein is known as secondary drying. Bound water molecules are too small to act as structural stabilizers the way sugars or other large molecules with polar surfaces can. Should bound water return it is likely to destabilize the hydrogen bonding to the sucrose or to neighboring protein molecules in the absence of sucrose and therefore increases the degrees of freedom of motion available. Once the bound water returns in the presence of moisture, the glass transition temperature is reduced. It is unclear why untreated samples performed better than treated samples when stored desiccated without sucrose (Figure 6). We found that reducing the temperature of storage always increases the activity of surface-bound protein after rehydration (Figure 9). However, for catalase, -20 °C gave better results than storage at 23 or 4 °C. Storage at room temperature or 4 °C gave similar results for catalase. Perhaps this implies that catalase is a protein of lower stability than HRP and requires a lower temperature for maintaining stability. Our results are in contrast to those of Seetharam (2006) who appeared to have found little advantage of cold storage. However they did find that a disaccharide (trehalose) was beneficial for freeze-drying microtubules on surfaces. However, after 5 days, storage motility had dropped. In fact, after 24 days of storage, less than half the microtubules moved. Uppalapati et al.18 were more successful, and found they could dry kinesins on glass surfaces, and this could increase their lifetime from days to more than 4 months when stored desiccated at 4 °C. Furthermore, critical point drying gave better results than freeze-drying. They appeared to be as successful as we have been in maintaining the function of a dry protein (kinesin) on a surface; however, their incubation solution contained casein and a detergent, which may help stabilize kinesin in a dry state, but these additives may cause problems with other protein assays. Dankwardt et al.4 were also successful in maintaining the activity of antibodies on microtiter plates if the antibody was postcoated with bovine serum albumin (BSA). After drying, the antibody activity was 40% of the control and 30% two weeks later. Better results were obtained if the plates were sealed after coating and washing and presumably stored wet. In this case, 100% of the activity was retained after 4 weeks storage at 4 °C. Our protocol requires the addition of sucrose only. Nath5 found that β-galactosidase on hydrogel-coated glass slides became completely inactive after 12 days stored dry at 4 °C. However the attachment of proteins involved in proteinprotein interaction studies could be safely spin-dried on these same surfaces; however, storage stability was not studied. They concluded that enzymes are much more sensitive to surface

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attachment than other proteins. In our work we could safely maintain the activity of HRP for 4 months in the dry state. Reduction of the unfolding rate at reduced temperature is expected on the basis of a two-state chemical reaction rate, for unfolding from the folded to the unfolded state. Such a reaction will be temperature activated and would be expected to obey an Arrhenius relation. The glass transition is expected to affect the activation energy for such a process, so the unfolding rate is expected to show a sudden increase above the glass transition temperature. The glass transition temperature of freeze-dried protein lies in the range 120-200 °C27 but is expected to be lower on surfaces. It is expected therefore that the unfolding rate relevant to this work is that applied below the Tg. By fitting an Arrhenius expression to the temperature dependence of the rate of function loss when the freeze-dried enzyme is rehydrated using Figure 9 data, we find a value of 60.4 kJ per mole for the activation energy. This value is larger than the value of 50.7 kJ per mole we find for HRP in solution (data from Figure 1). This shows that the barrier for unfolding in the freeze-dried state is larger than in solution.

5. Conclusion We have shown that it is possible to immobilize enzymes, using, as examples, HRP and catalase on a surface, freeze-drying them, and recovering the function of the surface-attached enzyme after rehydration. The extent to which the function is recovered is increased if four conditions are met. These are (1) the use of a polymer surface that has been PIII treated as the surface for attachment, (2) freeze-drying with sucrose in the buffer solution, (3) dry storage with desiccant after freezedrying, and (4) maintaining the freeze-dried protein at a reduced temperature. Other than sucrose, no other additives are needed to maintain the function of the protein in the dry state on our surface. Storage in the dried state removes the possibility of loss of function arising from any process that occurs as a result of interactions with water in solution. The possibility of longterm storage of freeze-dried proteins on surfaces in the dry state is now established, with many possible chemical, pharmaceutical, and medical applications. Acknowledgment. We gratefully acknowledge financial support from the Australian Research Council. Supporting Information Available. Stability of PE-attached HRP in phosphate buffer as a function of treatment time. This material is available free of charge via the Internet at http:// pubs.acs.org

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