Electrospray Deposition as a Method To Fabricate Functionally Active

in the electrospray deposition (ESD). It was shown that protein inactivation upon ESD is highly dependent on voltage and current used. Humidity and th...
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Anal. Chem. 1999, 71, 1415-1420

Electrospray Deposition as a Method To Fabricate Functionally Active Protein Films Victor N. Morozov*,† and Tamara Ya. Morozova†

W. M. Keck Foundation Laboratory for Biomolecular Imaging, Department of Chemistry, New York University, New York, New York 10003, and Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia

Electrospray ionization is a routine method in MS analysis of proteins and other biopolymers. Deposition of the electrospray products onto a conductive electrode is suggested here as a means to manufacture functionally active protein films. Recovery of the specific hydrolytic activity of the electrosprayed alkaline phosphatase (AP) was used as a probe for preservation of protein intactness in the electrospray deposition (ESD). It was shown that protein inactivation upon ESD is highly dependent on voltage and current used. Humidity and the presence of protective substances in solution also affect the process. Complete preservation of the enzyme activity was observed when the ESD was performed at low current and humidity in the presence of disaccharides. Deposition of proteins and other biospecific molecules onto an electrode surface is a common task in fabrication of enzyme electrodes and other types of biochemosensors.1,2 Known techniques include entrapment of protein molecules into a photopolymerizable gels,3,4 photolithography and lift-off technique,3-7 electroassisted deposition from solution,8,9 and ink-jet printing.10-11 The latter technique becomes advantageous when numerous * Corresponding author: (fax) 7-0967-73-0623; (e-mail) morozov@ pbc.iteb.serpukhov.su. † Present address: Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia. (1) Buerk, D. G. Biosensors. Theory and Applications; Technomic Publ. Co.: Lancaster, Basel, 1993. (2) Canh, M. Biosensors; Chapman & Hall: London, New York, 1993. (3) Koudelka-Hep, M.; de Rooij, N. F.; Strike, D. G. In Methods in Biotechnology, Immobilization of Enzymes and Cells; Bickerstaff, G. F., Ed.; Humana Press: Totowa, NJ, 1997; Vol. 1, Chapter 11. (4) Strike, D. J.; van den Berg, A.; de Rooij, N. F.; Koudelka-Hep, M. In Diagnostic Biosensor Polymers; Usmani, A. M., Akmal, N., Eds.; ACS Symposium Series 556; American Chemical Society: Washington, DC, 1994; Chapter 23. (5) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12287-12291. (6) Hengsakue, M.; Cass, A. E. G. Bioconjugate Chem. 1996, 7, 249-254. (7) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chem. 1995, 67, 36053607. (8) Johnson, K. W.; Allen, D. J.; Mastrototaro, J. J.; Morff, R. J.; Nevin, R. S. In Diagnostic Biosensor Polymers; Usmani, A. M., Akmal, N., Eds.; ACS Symposium Series 556; American Chemical Society: Washington, DC, 1994; Chapter 7. (9) Strike, D. J.; de Rooij, N. F.; Koudelka-Hep, M. In Methods in Biotechnology, Immobilization of Enzymes and Cells; Bickerstaff, G. F., Ed.; Humana Press: Totowa, NJ, 1997; Vol. 1, Chapter 12. (10) Newman, J. D.; Turner, A. P. F.; Marrazza, G. Anal. Chim. Acta 1992, 262, 13-17. 10.1021/ac9808775 CCC: $18.00 Published on Web 02/25/1999

© 1999 American Chemical Society

protein samples should be deposited without cross-contamination. It requires, however, a precise translation mechanism when spots of micrometer size are deposited. Ink-jet printing becomes slow when numerous multicomponent samples are manufactured since deposition of each spot is accompanied with a mechanical motion. Having in mind development of a simple method to simultaneously fabricate numerous protein microsamples from diluted solutions we came upon the idea to use electrospray deposition (ESD) for this purpose. ESD was first introduced in nuclear research to fabricate thin uniform radioactive sources12-14 in 50s. Other known applications of ESD include preparation of samples for field desorption ionization in MS,15,16 metal oxide films,17 polymer coating on electrodes,18 and modification of a silicon surface with a polypeptide to enhance cell adhesion.19 ESD was also used to prepare samples of DNA20 and protein molecules21 for imaging with a scanning tunneling microscope. To our best knowledge, nobody has ever tried to use ESD to prepare functionally active protein layers, though recent development of the ES technique as applied to MS is encouraging in this respect. It has been well established in MS studies that not only covalent bonds but also noncovalent interactions preexisting in solution between protein and ligand molecules can be preserved if ES is performed under mild conditions: (i) protein is sprayed from a nondenaturing solvent, (ii) the temperature of the drying gas is reduced, and (iii) the intensity of collisions with gas molecules on the way into the vacuum part of the mass spectrometer is low due to low orifice voltage. Numerous examples of observations of such noncovalent (11) Lemmo, A. V.; Fisher, J. T.; Geysen, H. M.; Rose, D. J. Anal. Chem. 1997, 69, 543-51. (12) Carswell, D. J.; Milsted, J. J. Nucl. Energy 1957, 4, 51-54. (13) Robinson, P. S. Nucl. Instrum. Methods 1966, 40, 136-140. (14) van der Eijk, W.; Oldenhof, M.; Zehner, W. Nucl. Instrum. Methods 1973, 112, 343-351. (15) Matsuo, T.; Matsuda, H.; Katakuse, I. Anal. Chem. 1979, 51, 1329-1331. (16) Murphy, R. C.; Clay, K. L.; Mathews, W. R. Anal. Chem. 1982, 54, 336338. (17) Chen, C.; Kelder, E. M.; van der Put, P. J. J. M.; Schoonman, J. J. Mater. Chem. 1996, 6, 765-771. (18) Hoyer, B.; Sørensen, G.; Jensen, N.; Nielsen, D. B.; Larsen, B. Anal. Chem. 1996, 68, 3840-3844. (19) Buchko, C. J.; Kozloff, K. M.; Sioshansi, A.; O’Shea, K. S.; Martin, D. C. In Mater. Res. Soc. Symp. Proc.; Cotell, C. M., Meyer, A. E., Gorbatkin, S. M., Grobe, G. L., III, Eds.; MRS: Pittsburgh, PA, 1996; Vol. 414, pp 23-28. (20) Thundat, T.; Warmack, R. J.; Allison, D. P.; Ferrel, T. L. Ultramicroscopy 1992, 42-44, 1083-1087. (21) Morozov, V. N.; Seeman, N. C.; Kallenbach, N. R. Scanning Microsc. 1993, 7, 757-779.

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complexes in ESI-MS, summarized in recent reviews,22,23 make us to believe that ES ionization neither destroys the compact native structure of protein molecules nor irreversibly inactivates them. It is not known, however, whether biological macromolecules can survive the impact with a target electrode upon ESD. Destruction of both protein ions and substrate surface is well documented in studies of collisions of accelerated protein ions with mica and graphite.24,25 Even in the absence of any accelerating potential on the target electrode, interaction of a protein ion with its mirror charge will result in an acceleration of the ion in the vicinity of any dielectric surface.25 The possible damaging effect of such an impact is difficult to predict. The authors are aware of only two attempts to experimentally address the problem. Nohmi and Fenn performed a chromatographic analysis of the ES deposited poly(ethylene oxide) and did not find any breakage in the polymer chains.26 On the contrary, Cheng et al., revealed numerous altered DNA molecules in the electrophoretic analysis of the plasmid DNA ES deposited onto a dry stainless steel electrode.27 No such alterations in the DNA structure were found in their experiments when the DNA was ES deposited into a buffer droplet. Thus, available data are too scanty and contradictory and an investigation is required to elucidate whether ESD can be used to fabricate functionally active protein deposits and to find conditions beneficial for preservation of protein activity in the ESD. We chose AP as an object for the investigation since this enzyme is well studied,28,29 readily available, and extremely rapid to allow measurements of enzyme activity with a submicrogram amount of the protein. The specific activity of this enzyme was used here as a probe to study how different parameters and factors operating in the ESD affect functional activity of protein deposit. EXPERIMENTAL SECTION Materials. Alkaline phosphatase from bovine intestinal mucosa, p-nitrophenyl phosphate (pNPP substrate tablet set, Sigma Fast), trehalose, and sucrose were purchased from Sigma. All other salts and buffer reagents were of analytical grade or purer. Design of Capillaries for ES. Three different designs of the ES capillaries have been tested throughout this study. The first design, shown in Figure 1A, is similar to the “nanoelectrospray ion source”30,31 with a silver layer plated on the external surface of a glass capillary instead of a gold one. Glass capillaries with sealed ends were first treated for 20 s in an electrodeless plasma discharge at a reduced pressure (0.01 Torr, discharge power of 10-20 W, in a Pyrex glass chamber, 0.25 L). After such a plasma (22) Przybylski, M.; Glocker, M. O. Angew. Chem., Int. Ed. Engl. 1996, 35, 806826. (23) Smith, R. D.; Bruce, J. E.; Wu, Q.; Lei, O. P. Chem. Soc. Rev. 1997, 26, 191-202. (24) Reimann, C. T.; Quist, A. P.; Kopniczky J.; Sunqvist, B. U. R. Nucl. Instrum. Methods Phys. Res. 1994, B88, 29-34. (25) Sullivan, P. A.; Axelsson, J.; Altmann, S.; Quist, A. P.; Sunqvist, B. U. R.; Reimann, C. T. J. Am. Soc. Mass Spectrom. 1996, 7, 329-341. (26) Nohmi, T.; Fenn, J. B. J. Am. Chem. Soc. 1992, 114, 3241-3246. (27) Cheng, X.; Camp, D. A., II; Wu, Q.; Bakhtiar, R.; Springer, D. L.; Morris, B. J.; Bruce, J. E.; Anderson, G. A.; Edmonds C. G.; Smith, R. D. Nucleic Acids Res. 1996, 24, 2183-2189. (28) McComb, R. B.; Bowers, G. N., Jr.; Rosen, S. Alkaline Phosphatase; Plenum Press: New York, 1979. (29) Coleman, J. E.; Gettins, P. In Advances in Enzymology; Meister, A., Ed.; Wiley & Sons Inc.: New York, London, 1983; pp 381-453. (30) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (31) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167180.

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Figure 1. Types of the capillaries used as ion sources in this study: (A) capillary with the external electrode; (B) capillary with the internal electrode; (C) capillary with a liquid bridge. (1) plastic tubing, (2) glass capillary coated with the silver layer, (3) contact wire, (4) stainless steel tube, (5) glass capillary, (6) internal tungsten or stainless steel electrode, (7) plastic capillary.

cleaning, they were activated in an acidified solution of SnCl2 (20 g of SnCl2 and 50 g of HCl per 1 L), washed with water, and covered with a silver using the “silver mirror” reaction.32 We will refer further to this design as to the capillary with the external electrode. The second design, shown in Figure 1B, is a modification of our ES capillary described recently.21 The metal electrode (tungsten or stainless steel wire) is not exposed to a gas phase in this design, thus reducing the risk of corona discharge at high voltage. We will further refer to this as to the capillary with the inner electrode. In the third version of the capillary design (bridge capillary), we tried to completely avoid any contact of the metal electrode with protein solution by introducing a liquid bridge between them. As seen in Figure 1C, the external surface of the stainless steel tube-4 is used as an electrode exposed to the interior of the remote end of the large external glass capillary-5. Since the diameter of the capillary-5 is more than 10 times larger than that of the inner plastic capillary-7, the conductivity of the former exceeds more than 100 times that of the inner capillary and most of the current flows through the large external capillary-5 filled with 10-4 M KCl solution, whereas the internal thin plastic capillary-7 is used to supply protein solution. A microprocessor-controlled syringe pump (Cole-Parmer) in combination with a 10-µL Hamilton microsyringe was used to feed the ES capillary. Flow rate was 6-9 µL/h. The outer diameter of the capillary tips varied in different experiments between 50 and 100 µm. Chamber with Controlled Humidity. To control humidity during ESD experiments and to protect protein samples from contamination with dust particles present in the ambient air, we performed the ESD in a small (0.5 L) nearly cubic acrylic box, schematically presented in Figure 2. A glass window was glued to one side of the box to enable good visibility of the ES plume and substrate under a stereomicroscope. Humidity and temperature inside the chamber were measured with solid-state digital (32) Yampolskii, A. M.; Il′in, V. A. Short Manual on Galvanic Technique; Mashinostroenie: Leningrad, 1981; Chapter 12.

Figure 2. Schematic of the chamber used to deposit protein on the quartz microbalance. See text for more details.

sensors (Fisher’s product, 2-4% accuracy for humidity and 0.2 °C accuracy for temperature). To increase the rate of sensor response and to keep the same humidity in all parts of the chamber, the air was stirred with a small fan. Dry air from a tank or that bubbled through water was introduced into the chamber until the required humidity was reached. After that, the fan was stopped and ES started. Occasionally portions of dry or wet gas were added during ESD, if the humidity deviated by more than 3-5% from the required one. Safety Considerations. To avoid occasional electric shock in handling the high voltage used here, it is advisable to connect the capillary with a high-voltage power supply via a resistor of 10-100 MΩ. Measurements of Mass of the ES-Deposited Protein. A home-built quartz crystal microbalance33 (QCM) was used to measure the mass of the ES deposited protein. The microbalance was made of the commercial AT-cut quartz crystals (12-17 MHz, with silver electrodes of 5 mm in diameter) after removal of their protective shells. QCM calibration was performed using 0.1% solution of sugar in water. A microdroplet of this solution (0.251.0 µL) was placed in the middle of the quartz electrode using the microsyringe pump and dried as a spot of 1-1.5 mm in diameter. The quartz was placed in a closed drying chamber of the QCM, connected to the oscillator circuit and dried in a flow of dry nitrogen or air until constant frequency was reached. The difference between the resonance frequency of the clean quartz crystal and that of the same crystal loaded with the sugar spot was calculated for every sugar mass. Calibration curves were linear in the range of 0-2 µg. Root-mean-square deviations of the experimental points from the linear regression line were within 2-3%. To diminish the scatter of experimental points, the deposition should be made in the center of the quartz electrode, since the electrode periphery is less sensitive to mass changes.33 ESD of protein was performed on the QCM electrode as shown schematically in Figure 2. The electrode was grounded, and a plastic mask made of a Teflon or Parafilm sheet was superimposed (33) Sauerbrey, G. Z. Phys. 1959, 155, 206-222.

on the quartz crystal in such a way as to position a round hole in the mask over the center of the electrode. After deposition, the protein sample was dried in the drying chamber of the QCM until constant frequency was reached. The dry mass of the protein deposit was calculated from the shift in the quartz resonance frequency using the calibration curve. Measurements of Specific Activity of the ES-Deposited AP Samples. A commercial dry powder of AP was dissolved in water in a concentration of ∼1 mg/mL, dialyzed overnight against a solution of 10-5 M MgCl2, 10-5 M ZnCl2, pH 7-8, poured into Eppendorf tubes, centrifuged at 3000g for 1-2 min, and kept at -20 °C. Before the experiment, an aliquot of the stock solution was unfrozen, diluted 5 times with water, and centrifuged again. ES capillary, tubing, and microsyringe were filled with the protein solution. No bubbles were allowed to penetrate the system. Typically, 1 µL of the protein solution with a concentration of 0.15-0.20 mg/mL was electrosprayed in each deposition. After the dry mass of the deposit was measured as described above, the protein spot was dissolved in 40 µL of a buffer solution (0.2 M TRIS/HCl buffer, pH 9.5, 1 mM MgCl2, 0.1% Tween 20). AP activity was measured immediately after spot extraction by adding 2-20 µL of the extract to 1.0 mL of the pNPP solution in the TRIS/HCl buffer prepared from Sigma Fast tablets. Measurements were performed in a thermostated room, at temperature of 25 ( 1 °C. Activity, determined by linear regression analysis of absorbance measurements at 410 nm recorded for 2-30 min with a computer-controlled spectrophotometer (AVIV, model 118DS), exhibited linear dependence upon the enzyme concentration. Specific enzyme activity was calculated using protein mass measured as described in the previous section. Every series of the deposition experiments was accompanied by control measurements of specific AP activity in the solution used for the ESD and in samples obtained by direct drying. In the latter case 1 µL of the AP solution was applied directly onto the quartz electrode and dried in a flow of air. After measurement of its dry mass, the deposit was dissolved and the specific AP activity was measured as described for the ES-deposited samples. Effect of Carbohydrates. To study the effect of sucrose and trehalose on the recovery of AP activity after drying, microdroplets (5 µL) of the diluted AP solutions were placed onto a glass surface and equal volumes of water or water solution containing different amounts of carbohydrates were added to each droplet. Well-mixed droplets were dried in a desiccator under a reduced pressure created with a water pump. Dry spots were then dissolved, and specific AP activity was measured as described for the ESdeposited spots. In the ESD experiments with carbohydrates, protein concentration in the extract was calculated by assuming that the proteinto-carbohydrate ratio is the same in the solution and in the ES deposit. RESULTS AND DISCUSSION Measurements of Mass of the ES-Deposited Samples. The mass of the deposit is determined from the shift in the resonant frequency of the quartz oscillations. However, this shift may depend not only on the deposited mass but also on the viscoelastic properties of the deposit. It seems that protein samples prepared by ESD under various humidities have very different internal structure and packing density: they are opaque or opalescent Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

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when prepared at humidity, A < 50%, and transparent, quite invisible (similar to those obtained upon direct drying), when ESdeposited under humidity A > 70%. To check the possible effect of such structural difference on the QCM measurements, special experiments were performed. After measurement of the quartz resonance frequency with dry ES deposit prepared at low humidity, the quartz was placed for 30-60 s in a Petri dish with A ) 100% and then dried again and its resonant frequency remeasured. The opalescent deposit turned into a transparent film as a result of such treatment. However, no changes in the resonance frequency accompanied this transformation of the deposit structure. We may, thus, conclude that variations in the structure of the ES deposit prepared under different humidities do not affect our mass measurements. Usually the mass of the deposit varied in the range of 0.020.3 µg, and QCM seems to be the only method capable of measuring such a small quantity of protein reliably. Conventional methods, like Lawry’s and BCA, require at least 0.5-1 µg of protein.34 Of course, specific activity defined per unit of mass of the dry residue is underestimated since the residue consists not only of the protein exclusively. In those conditions, comparison of the specific activity in the deposit with that in the solution used for ESD is possible only when the latter is also related to the dry mass of the residue. That is why the same QCM method was used to measure protein quantity not only in the ES deposited and dried samples but in solution too. Furthermore, only the ratio of specific activities in dried or ES-deposited samples to those in the AP solution was used throughout this study. Provided the composition of the dry residue in the ES deposit and after direct drying of the same solution is similar, such a ratio should not depend on the presence of impurities in the solution. Actually the dry residue in our experiments consists mostly of protein, since the dialyzed protein solution was dried in all the mass measurements (e.g., 10-5 M ZnCl2 and MgCl2 salts in the 1 mg/mL protein stock solution contributes only 0.23% to the dry mass of the residue). ESD Efficiency. Defined as a ratio of the dry mass of the ES deposit to the total mass of protein in the electrosparyed volume of solution, this value shows a marked dependence on the diameter of hole in the mask, d, through which the sample was deposited, on the distance, h, from the capillary tip to the quartz electrode, on voltage, V, used in the ESD and on design of the ES capillary. The efficiency enhances with the increase in the hole diameter and voltage and with decrease in the h. With typical geometrical parameters, h ) 10-15 mm, d ) 2 mm, V ) +(3-4) kV, ESD efficiency varied between 60 and 80% depending on the capillary design, as it is seen from Table 1. Slightly lower efficiency of the bridge-type capillary can be explained by a protein leakage into the liquid bridge. AP molecules are negatively charged35 at pH >4.4 and part of them can migrate through the bridge to the positively charged capillary electrode. The efficiency of the ESD from the capillary with the internal electrode did not show any notable dependence on humidity: averaged over 48 independent measurements under humidity varying between 10 and 90% the ESD efficiency was 74 ( 6% with (34) Bollag, D. M.; Rozycki, M. D.; Edelstein, S. J. Protein Methods; John Wiley & Sons Inc.: Publ. New York, 1996; Chapter 3. (35) Engstro¨m, L. Biochim. Biophys. Acta 1961, 52, 36-41.

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Table 1. Efficiency and Percentage of Recovery of Specific AP Activity upon Electrospray Deposition from Different Capillaries under Identical Conditionsa

activity,b % efficiency,c %

internal electrode

external electrode

bridge type

55 ( 10 79 ( 7

32 ( 9 78 ( 8

31 ( 12 62 ( 9

a Voltage at the capillary electrode, +3-4 kV; current, 5-50 nA; solution flow rate, 0.1 µL/min; distance between the capillary tip and quartz surface, 10 mm; humidity in the ES chamber, 65 ( 5%. b Ratio of the specific activity of the ES-deposited AP to that in the initial solution. c Ratio of mass of the ES-deposited sample to the mass of protein in the electrosprayed solution volume.

V ) 3-4 kV. Voltage increase to 6-7 kV resulted in increase of the ESD efficiency up to 100%. Specific Activity of the ES-Deposited AP. Expressed as a percent ratio of specific AP activity in the extract of the ES deposit to that in the initial solution, this characteristic is used here as a measure of preservation of native properties of protein in ESD. As one can see from the data presented in Figure 3A and B, AP is subjected to the highest damage and loses nearly all its activity when ESD is performed under large voltage (V > 7-8 kV) and current (I > 500-1500 nA). The following factors may be thought of as contributing to the AP inactivation under these conditions: (i) inactivation inside the ES capillary due to the electrochemical reactions on electrode, (ii) inactivation as a result of reaction with corona products in the gas phase, (iii) inactivation as a result of impact with the target electrode, and (iv) inactivation as a result of drying. Drying upon the ESD is expected to have an effect similar to that in direct drying and to be independent of the current and capillary design. As seen from Figures 3A, 4, and 5, AP is really inactivated to the same extent (by ∼45%) upon direct drying on a glass surface in a vacuum, upon drying in air flow on the silver electrode of QCM, and upon ESD from the capillary with the internal electrode at humidity A ) 65% and currents not exceeding 50 nA. Thus, one may conclude that under these mild ESD conditions no other damaging factors operate besides the drying itself. Under similar currents and humidity, AP was notably more inactivated (by 70%) when ES deposited from two other types of ES capillaries (see Figure 3B and Table 1). The superiority of the capillary with the internal electrode over the one with the liquid bridge appears surprising and unexplainable if the electrochemical reaction is assumed to be the main damaging factor in the ESD. As seen from comparison of the lower curves in parts A and B of Figure 3, the capillary with the internal electrode is notably less damaging than that with the external one under all voltages and currents. This also contradicts the hypothesis about protein denaturation by products of the electrochemical reactions on the electrode. To further check the hypothesis, we measured and compared specific activities of AP deposited from the same capillary at low current before and after ESD with a current of 1500 nA. It was expected that accumulation inside the capillary of oxidized or otherwise damaged AP molecules or accumulation of electrochemical products harmful for the AP activity will result in a diminished activity of the successive ES deposit. Bovine intestinal AP is known to be irreversibly inactivated28 by exposure to pH below 4.5-5.0 and estimates of van Berkel et al. show36 that pH in the ES capillary can drop up

Figure 3. Effect of current on recovery of the specific activity of the ES-deposited AP in the absence (empty circles) and in the presence of sucrose (filled circles). Sucrose was added in the amount corresponding to 50% of the dry weight of protein. (A) capillary with the internal electrode; (B) that with the external one. Ordinate is the specific activity of the ES-deposited AP related to that in the initial solution. ESD was performed at the relative humidity of 65 ( 5% and flow rate of solution of 6.0 µL/h.

to pH 3.5 during the ES with current I ) 650 nA and flow rate of 1.25 µL/min. However, no notable difference between the specific AP activities in the preceding and successive deposits was found for the capillary with the internal electrode and for that with the external one. We believe, therefore, that inactivation of AP at high voltages and currents occurs not inside but outside the capillary as a result of reactions with active products of corona discharge or due to the impact with the target electrode. The impact energy should grow with increase of potential on the capillary. This may explain the enlarged inactivation of AP at high voltage and current. Generation of corona products in the ESD at I > 200-300 nA was apparent from the characteristic smell of ozone in the ES chamber. Maintenance of high current even after the microsyringe pump was switched off and visible ES plume disappeared was also indicative for the presence of corona discharge under these conditions. It was also noted that ES at high current resulted in removal of the Ag plating from the capillary with the external electrode. This capillary supports corona discharge more readily than that with the internal electrode, explaining why AP is inactivated at lower currents and to a higher extent when ES deposited from the former capillary. It is noteworthy that contamination of the AP deposit with the Ag ions from the Ag plating of the capillary and from the Ag electrode of the QCM seems not to contribute to the AP inactivation, since addition of 10-4 M AgNO3 to AP solution does not cause any changes in the AP activity. Whatever the mechanism of inactivation in ESD at large voltages and currents is, the most important result of this work consists of establishing the fact that ES-deposited AP preserves as much of its activity as during direct drying provided the ESD is performed from a capillary with the internal electrode at a voltage not exceeding +4.5 kV and a current less than 50 nA. One may conclude that under these conditions all damaging factors listed above do not work and the drying process itself seems to remain the only inactivating factor left. It has been well documented that many proteins notably lose their activity when subjected to freeze-drying and some of them (36) van Berkel, G. J.; Zhou, F.; Aranson, J. T.; Int. J. Mass Spectrom. Ion Processes 1997, 162, 55-67.

Figure 4. Effect of carbohydrates on the recovery of the AP activity after direct drying. Empty and filled circles for sucrose and trehalose, respectively. Activity is related to that in the initial solution.

were found to be completely inactivated upon freeze-drying or drying at room temperature.37,38 It is also well-known that disaccharides are capable of protecting proteins upon drying.38 Data presented in Figure 4 indicate that AP is equally well protected from such inactivation by both sucrose and trehalose. Addition of 0.5 g of the disaccharide/1 g of dry protein is enough to preserve 100% of the AP activity. The upper curve in Figure 3A shows that the same substance, sucrose, that protects AP activity upon drying equally well protects it upon ESD. Taken at the same concentration that preserves 100% of AP activity upon drying (0.5 g/g of dry protein), sucrose also increases up to 100% the recovery of the AP activity in the ES deposit, provided the deposition was performed at low current and from the capillary with the internal electrode. These data provide an additional support in favor of the idea that drying becomes the main damaging factor in the (37) Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Aurell Wistrom, C. Biochem. J. 1987, 242, 1-10. (38) Gibson, T. D.; Woodward, J. R. In Biosensors and Chemical Sensors; ACS Symposium Series 487; Edelman, P. G., Wang, J., Eds.; American Chemical Society: Washington, DC, 1992; pp 40-55.

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multiple cycles of wetting/drying accompanying landing of ES microdroplets which cannot completely evaporate on their way to substrate under this condition.

Figure 5. Effect of humidity on the recovery of the specific AP activity after ESD. Specific activity of the ES-deposited samples is related to that in the initial solution. ESD conditions: +4 kV at the internal electrode, current, 10-50 nA, and flow rate 6.0 µL/h.

ESD under such mild conditions. Data presented in Figure 5 also support this point of view. Rapid drying of the ES-generated droplets in the gas phase under low humidity is seen to be even less damaging for the AP activity (30% lost) than slow drying of the AP solution on a glass surface (45% lost as seen from Figure 4). However, the enzyme is subjected to larger damage if the ESD is performed at humidity higher than 65% presumably due to

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CONCLUSIONS We have shown in this paper that functional activity of AP can completely survive the electrospray deposition on a metal electrode if deposition is performed under mild conditions in the presence of protective substances. If further studies with other proteins having different pI, stability, and redeox properties (for example, those having freely accessible SH groups vital for enzyme activity) show that effective deposition is possible with many proteins, a background will be created for a new technology to fabricate biospecific elements of sensors, libraries, and diagnostic assays. Abbreviations: AP alkaline phosphatase, ES electrospray, ESD electrospray deposition, pNPP p-nitrophenyl phosphate, MS mass spectrometry, QCM quartz crystal microbalance. ACKNOWLEDGMENT We gratefully acknowledge the National Science Foundation (Grant BIR-9513571) and S.T. Research, Ltd. (Tokyo, Japan) for their financial support of this work. Received for review August 5, 1998. Accepted December 17, 1998. AC9808775