Anal. Chem. 1999, 71, 3110-3117
Electrospray Deposition as a Method for Mass Fabrication of Mono- and Multicomponent Microarrays of Biological and Biologically Active Substances 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, Russia 142292
Electrospray of protein and DNA solutions is currently used to generate ions for mass spectrometric analysis of these molecules. Deposition of charged electrospray products on certain areas of a substrate under control of electrostatic forces is suggested here as a method for fabrication of multiple deposits of any size and form. For example, multiple dots of protein, DNA, or other organic substances can be deposited simultaneously through an array of holes in a dielectric mask covering any slightly conductive substrate (membrane, wet glass, semiconductor, etc.). If every new substance is deposited after a shift of the mask with respect to the substrate, a multicomponent matrix is created under each hole. It is demonstrated that dots as small as 2-6 microns can be fabricated by such an electrospray deposition (ESD). It is also demonstrated that the ES-deposited proteins and DNA retain their ability to specifically bind antibodies and matching DNA probes, respectively, enabling use of the ESD fabricated matrixes in Dot Immuno-Binding (DIB) and in DNA hybridization assays. Modern genetic, clinical, and pharmaceutical analyses and monitoring of environmental pollution require methods enabling parallel tests of many compounds. The microarray industry is now a rapidly emerging area with many different methods and approaches exploited.1 Multicomponent test systems can be manufactured either by a photochemically controlled synthesis directly on a substrate2,3 or by delivery and deposition of presynthesized and purified substances onto the substrate. Thus, multiple dots of oligonucleotides and proteins were deposited by hand4 or * Corresponding author. Fax: 7-967-790-553. E-mail:
[email protected]. † Present address: Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Moscow Region, Russia 142292. (1) Shena, M.; Heller, R. A.; Theriault, T. P.; Konrad, K.; Lachenmeier, E.; Davis, R. W. TIBTECH 1998, 16, 301-306. (2) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (3) Fodor, S. P. A.; Rava, R. P.; Huang, X. C.; Pease, A. C.; Holmes, C. P.; Adams, C. L. Nature (London) 1993, 364, 555-556. (4) Guschin, D.; Yershov, G.; Zaslavsky, A.; Gemmell, A.; Shik, V.; Proudnikov, D.; Mirzabekov, A. Anal. Biochem. 1997, 250, 203-211.
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by using a robotic micromanipulation system.5 The latest development of such a delivery technique includes adaptation of ink jetting for deposition of biomolecules.6-8 The fundamental limitation of all these delivery techniques consists in a relatively low rate of the deposition since only one or a few dots are deposited at time. The recently developed stamp microcontact printing technique9,10 is free from such a limitation. It allows simultaneous deposition of many identical dots and has a remarkable spatial resolution. However, low efficiency of substance transfer, a necessity for a new stamp for each substance deposited or a thorough washing of the same stamp after deposition of each substance can be considered drawbacks of this method. To overcome the above-mentioned limitations we suggest here using the electrospray deposition (ESD) technique for simultaneous fabrication of numerous chips and libraries of proteins, DNA, and other biologically active compounds. In the ESD technique, solutions of these substances are electrosprayed, and charged electrospray products are deposited onto specified areas of a conductive substrate under control of electrostatic forces. ESD has been first used to produce thin radioactive sources in nuclear physics.11-14 Other known applications of ESD include formation of layers of semiconductive ceramics,15 formation of a protective polymer layer on an electrode surface,16 preparation of samples for MS studies,17,18 and modification of a silicon surface (5) Shalon, D.; Smith, S. J.; Brown, P. O. Genome Res. 1996, 6, 639-645. (6) Newman, J. D.; Turner, A. P. F. Anal. Chim. Acta 1992, 262, 13-17. (7) Blanchard, A. P.; Kaiser, R. G.; Hood, L. E. Biosens. Bioelectron. 1996, 67, 687-690. (8) Lemmo, A. V.; Fisher, J. T.; Geysen, H. M.; Ross, D. J. Anal. Chem. 1997, 69, 543-551. (9) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225-2229. (10) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3972-3975. (11) Carswell, D. J.; Milsted, H. J. Nucl. Energy 1957, 4, 51-54. (12) Lauer, K. F.; Verdingh, V. Nucl. Instrum. Methods 1963, 21, 161-166. (13) Loventhal, G. C.; Wyllie, H. A. Nucl. Instrum. Methods 1973, 112, 353357. (14) van der Eijk, W.; Oldenhof, W.; Zehner, W. Nucl. Instrum. Methods 1973, 112, 343-351. (15) Chen, C.; Kelder, E. M.; van der Put, P. J. J. M.; Schoonman, J. J. Mater. Chem. 1996, 6, 765-771. (16) Hoyer, B.; Sorensen, G.; Jensen, N.; Nielsen, D. B.; Larsen, B. Anal. Chem. 1996, 68, 3840-3844. (17) Matsuo, T.; Matsuda, H.; Katakuse, I. Anal. Chem. 1979, 51, 1329-1331. 10.1021/ac981412h CCC: $18.00
© 1999 American Chemical Society Published on Web 07/02/1999
with a layer of silk-forming peptides19 to promote adhesion of living cells. ESD has also been occasionally used to prepare samples of DNA,20 proteins,21 and synthetic polymer molecules22 for imaging with scanning tunneling and atomic force microscopy. The authors are aware of only one publication describing use of ESD for preparation of a microarray of dots of a silklike polypeptide on a silicon substrate.19 The microarray was fabricated by ESD through a conductive shadow mask. However, microarrays of proteins, DNA, or organic substances have never been produced by the ESD method. A central problem in the ESD of proteins and DNA consists in preserving functional properties of these molecules upon electrospray and upon subsequent impact of the charged ES products with a substrate surface. We have recently shown23 that under properly chosen conditions specific catalytic activity of the ES-deposited alkaline phosphatase is completely preserved. In this paper we give additional evidence for preservation of functional properties of the ES-deposited biological molecules, such as antigenic and catalytic properties of proteins and hybridization ability of DNA molecules. EXPERIMENTAL SECTION Reagents. λ DNA and a mixture of biotinilated BstE II restriction fragments of λ DNA were purchased from the Biolabs Co. (Beverly, MA). Dyes were from a set purchased from the Chem. Services Co. (West Chester, PA). All the other chemicals (of analytical grade or purer) were purchased from Sigma Chemical Co. (St. Louis, MO). Principle of Microarray Fabrication. As schematically illustrated in Figs. 1A and 1B, numerous local deposits (dots, spots) can be produced under control of a local electrostatic field attracting charged ES products to specified substrate areas. One way to form such a field is to cover a conducting substrate with a dielectric mask containing an array of holes, as shown in Figure 1A. The electric field in this case is a sum of an attracting field protruding through the holes and a repelling field of the mask charged with a small amount of the adsorbed ES products. Another way to form a similar local electrostatic field is to locally increase conductivity of a photoconductive insulator layer (used as a substrate) by illuminating it through an appropriate photomask. This will result in deposition of the charged ES products into the illuminated areas, as illustrated in Figure 1B. In addition to light, any other physical factor (temperature, radiation, injection of charges,24,25 etc.) capable of increasing a local electric conductivity of a dielectric film can be exploited in ESD to control the spatial position of dots on the substrate. The array of dots formed in such a way is monocomponent. The number of dots corresponds to the number of holes in the (18) Murphy, R. C.; Clay, K. L.; Mathews, W. R. Anal. Chem. 1982, 54, 336338. (19) Buchko, C. J.; Kozloff, K. M.; Sioshansi, A.; O’Shea, K. S.; Martin, D. C. Mater. Res. Soc. Symp. Proc. 1996, 414, 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.; Kallenbach, N. R. Scanning Microsc. 1993, 7, 757-779. (22) Morozov, V. N.; Morozova, T. Ya.; Kallenbach, N. R. Int. J. Mass Spectrom. Ion Processes 1998, 178, 143-159. (23) Morozov, V.; Morozova, T. Ya. Anal. Chem. 1999, 71, 1415-1420. (24) Reiser, A.; Lock, M. W. B.; Knight, J. Trans. Faraday Soc. 1969, 65, 21682185. (25) Kallmann, H.; Pope, M. J. Chem. Phys. 1960, 32, 300-301.
Figure 1. Schematic of parallel fabrication of multicomponent matrixes by electrospray deposition. (A) Deposition through an array of holes in a dielectric mask. (B) Deposition onto illuminated areas of a photoconductive layer. After deposition of each compound, the dielectric mask or photomask is shifted to expose new areas of substrate for deposition of the next compound.
mask. To produce multicomponent matrixes and libraries, such a deposition should be repeated for every new substance after a small shift of the substrate with respect to the dielectric or a photomask. Such a displacement should be larger than the size of the dots but smaller than the distance between the neighboring holes in the mask. After deposition of all the substances a multicomponent matrix is formed under each hole. In total, as many identical multicomponent matrixes are formed in parallel as there are holes in the mask. Arrays of addressable electrodes may provide an alternative to use of a dielectric or photomask. Applying an attracting potential to certain electrodes and a repelling one to the rest of them will result in a preferred deposition of the charged ES products onto the former electrodes. Although this method looks similar to the known electrophoretic deposition of proteins from solution,26 use of ESD is advantageous since the lack of a direct contact with protein solutions prevents adsorption of solutes onto the electrodes, thus decreasing the cross-contamination of the deposits. (26) Johnson, K. W.; Allen, D. J.; Mastrototaro, J. J.; Morff, R. J.; Nevin, R. S. Chapter 7. In Diagnostic Biosensor Polymers; Usmani, A. M., Akmal, E. N., Eds.; ACS Symposium Series 556; American Chemical Society: Washington, DC, 1994.
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The ESD methods suggested here are essentially more efficient as compared with the ESD method using a shadow mask.19 In the latter method, deposition is not under the control of local electrostatic forces and, therefore, occurs uniformly on the substrate and on the shadow mask itself with a large waste of valuable materials. Apparatus. Different designs of the ESD device have been used throughout this study. All basically included (i) a glass capillary with a hydrophobic external surface and an internal platinum electrode21-23 of 50 micrometers in diameter, (ii) a flat dielectric mask with an array of holes, (iii) a flat conducting substrate, and (iv) a glovebox or a plastic chamber23 to control gas composition and to protect the deposit from contamination with dust particles present in the ambient air. The latest version of the device included a guard ring kept under a potential of the capillary and a perforated dielectric shield to increase the efficiency of deposition (see Figs. 1A and 1B). Displacement of mask vs substrate was performed either manually or by using simple accessories, including a homemade dual axis translation stage controlled with a pair of micrometer screws. In the latter case the substrate was glued or sucked to a micropositioner table, whereas the mask was rigidly fixed to a basement. First, ESD experiments were performed under a constant flow rate maintained with a microsyringe pump. The trouble with use of the pump is that occasional droplets are ejected from the ES capillary upon any instability in the ES, e.g., upon the passage of a small dust particle through the tip. Subsequently, we abandoned the pump in favor of a free capillary, where the delivery of solution is automatically regulated by the spray itself. To increase the ESD rate, a pressure of 3-30 cm of water was applied to the capillary interior. ES was controlled with a low-power stereomicroscope. The ES plume was illuminated with a beam of a 5 mW He/Ne laser. Masks made of different inorganic (mica, glass) and plastic materials (polypropylene, polyethylene, Teflon, polystyrene, and others) were tested. Arrays of holes, 0.4-1.5 mm in diameter, were cut in the plastic masks with punches. Holes in mica were drilled with a conical tungsten wire 0.4 mm in diameter. In some cases woven polypropylene or nylon meshes (see Figure 2A) were used as masks. These were purchased from the Small Parts Co. (Miami, FLA). Several materials have been used as substrates: metals (aluminum plating on a plastic film, gold on mica and silicon), semiconductors (SnO2 layer on glass), nitrocellulose (Micronseparation, Inc.), and PVDF membranes (Immobilon-P, Millipore Co.), Whatman paper, plasma-cleaned glass, and freshly cleaved mica. Weakly conducting substrates such as Whatman paper or membranes can be used both in dump and in dry states. Dump membrane can be directly placed on a metal electrode. To ensure a reliable electric contact with dry membrane, the opposite surface of the membrane was covered with a conducting carbon paste. Our experiments indicated that ESD could be performed on such a membrane even in dry air. A photoconductive dielectric layer on an aluminum cylinder from a Xerox machine was used as a substrate in the experiment on the photo ESD. The layer was illuminated by a diffraction pattern produced by the beam of a 5 mW He/Ne laser passed through a nylon mesh. 3112 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999
ESD was performed with the positive potential on the capillary in all cases. Safety Considerations. To avoid occasional electric shock in handling from high voltage used here it is advisable to connect the ES capillary with a high-voltage power supply via a resistor of 10-100 MOhm. Methods. Deposition of Dyes. Dyes were deposited on a dry nitrocellulose membrane from 0.01 to 1.0% solutions in water, EtOH, or MeOH from a capillary with an internal diameter of tip 30-50 µm. Slight pressure (3-8 cm of water) was applied to dye solutions to increase the rate of solution flow to 1-2 µL/min. Typically 1 µL of each dye solution was deposited. Deposition of Proteins. Proteins (bovine intestinal AP, horseradish peroxidase, HHb, HSA, BSA, OA, the IgG fraction of a goat anti-HSA antiserum, mouse monoclonal antibiotin AP conjugate) were deposited from water solutions with concentrations 0.1-1.0 mg/mL. The solutions were dialyzed for 12-24 h before ESD to reduce their electric conductivity. It was found to be difficult to obtain a stable ES of solutions with conductivities exceeding 200300 µS cm-1. Only AP solution, 1 mg/mL, was dialyzed overnight against a solution of 10-5 M MgCl2/10-5 M ZnCl2, pH ) 7-8. This solution was centrifuged at 3000g for 1-2 min, diluted 10fold, and ES-deposited. Peroxidase was sprayed from a water solution containing 1 mg/mL of peroxidase and 5 mg/mL of BSA. The amount of protein deposited in different experiments varied from 0.2 pg to 50 ng per dot as it was estimated from the overall volume of sprayed solution distributed among all the dots and from direct measurements of spot volume with the AFM or Linnik’s microinterferometer. Typical experimental conditions for the deposition of proteins were: voltage, +(3.0-4.0) kV; current, 5-50 nA; humidity, 30-70%; flow rate, 0.1-0.2 µL/min. ESD Efficiency. Qualitative analysis of efficiency of the ESD through different dielectric masks was performed using Bromphenol Blue dye. This dye was deposited from 1% ethanol solution onto an optically flat Al surface. The deposits were then washed from the mask and the substrate with 1 mL of water each, and the dye concentration in the solutions was determined spectrophotometrically (at λ ) 589 nm) using the calibration curve obtained by a direct addition of the dye solution to water. AFM Imaging of the ES Deposits. Nanoscope II (Digital Instruments, Santa Barbara, CA) equipped with a 9-micrometer scanning head was used to visualize the ES-deposited dots on a gold-plated mica prepared as described in ref 21. Imaging was performed in the contact mode under dry nitrogen. A detailed description of the instrument calibration and imaging procedure is presented in our recent paper.22 Detection of Enzyme Activity in the ES Deposits. After deposition the membranes were placed in solutions of substrates producing insoluble colored products of enzyme reactions. 3,3′-Diaminobenzidine for peroxidase and 5-bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazolium for AP were used as substrates. Both substrates were purchased as kits from Sigma (Sigma Fast BCIP/ NBT and Sigma Fast DAB tablets, respectively) and used as recommended by the manufacturer. Dot Immuno-Binding (DIB) with the ES-Deposited Matrixes. (i) Indirect Immunoassay. Immobilon-P membrane was used as a substrate in these experiments. The membrane was soaked with
Figure 2. Examples of ESD through different masks onto different substrates. (A) Image of a polypropylene mesh used as a mask in some ES depositions. Distance between holes is 50 µm. (B) Mask made of an unidentified plastic with holes spaced 1.0 mm apart. (C) Dots of light green ES deposited on a SnO2-covered glass through the mask, shown in Figure 2A. (D) Dots of horse myoglobin deposited on mica through the same mask. The mask was directly placed on mica. The electrode contacted the mica surface via a slightly wet O-ring made of Whatman paper.22 (E) AFM image of a typical dot of the light green dye ES deposited on a gold layer through the mask shown in Figure 2A. Bar size is 1 µm. Height of the dot is 0.1 µm. (F) Array of the Br phenol blue dots ES deposited onto a photoconductive layer. Distance between the dots is 3 mm.
methanol and washed with deionized and distilled water. The wet membrane was placed on a polished Al table of a micropositioner. Four different antigens (HSA, BSA, HHb, and OA) were deposited as dots 100 µm in diameter containing approximately 10 ng of protein each. The membrane was then dried for 15 min at 37 °C, blocked for 1-1.5 h in PBS (10 mM Na phosphate buffer, pH ) 7.2, 0.9% NaCl) containing 0.05% Tween-20 and 1% of OA, and soaked in a solution of the goat anti-HSA antibodies (Sigma product, 40 µg/mL) for 1 h. It was then rinsed twice with PBS and reacted with rabbit antigoat IgG antibodies labeled with AP (Sigma product diluted to 1:2500). After washing off the excess of the antibodies with PBS, the position of the enzyme-labeled spots of HSA was detected by incubating the membrane in the
solution of the BCIP/NBT substrate of AP. To detect BSA spots, the same procedure was used but with rabbit anti-BSA antibodies (Sigma product, 70 µg/mL) and with the goat antirabbit IgG antibodies labeled with AP (Sigma product, diluted to 1:2500). (ii) Direct Immunoassay. Microarrays of the goat anti-HSA antibodies were fabricated by ESD of a 0.4 mg/mL water solution of them on wet Immobilon-P and nitrocellulose membranes at a voltage 4.0-5.0 kV, current 10-40 nA, and humidity 10-70%, in different experiments. Spots with diameters varying from 1 mm to 2-6 µm were fabricated using different masks. After blocking and reaction with the rabbit antigoat IgG AP conjugate (Sigma product diluted to 1:3000), the microarrays were colored by insoluble products of the AP reaction as described above. Analytical Chemistry, Vol. 71, No. 15, August 1, 1999
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ESD of DNA and Hybridization. (i) ESD on Membranes was performed with a solution of 0.2 mg/mL of λ DNA in 20% glycerol. The solution was boiled for 5 min and snap cooled on ice immediately before being placed into the ES capillary. The deposition was performed at 60% humidity on Gene Screen membrane (Du Pont Co.) soaked in 1 × SSC buffer. Two microliters of the DNA solution was electrosprayed at +(4.24.5) kV with a current of 20-30 nA. After the ESD, DNA was baked for 20 min at 75 °C, denatured in a solution of 0.5 M NaOH/ 1.5 M NaCl for 5 min, washed in 0.5 M TRIS-HCl buffer/1.5 M NaCl, pH ) 7.4, and UV cross-linked for 15 min. The membranes were then prehybridized for 1 h at 42 °C in 6 × SSC buffer, containing 45% formamide, 1% SDS, 5% dextran sulfate, 5 × Denhardt’s solution, and 100 µg/mL of denatured sonicated DNA from salmon testes. Hybridization was performed at the same temperature for 14 h in the same buffer to which the biotinilated λ-DNA probe was added for a concentration of 200 ng/mL. Hybridization was followed by washing at room temperature first with 2 × SSC buffer (5 min), then twice (5 min) with 2 × SSC/ 0.2% SDS solution, and finally with 0.1 × SSC/0.2% SDS at 62 °C twice for 15 min each. After being washed, the membranes were rinsed with the AP buffer (0.1 M TRIS-HCl, pH ) 7.5, 0.1 M NaCl, 2 mM MgCl2, 0.5% Tween-20), blocked with a mixture of 2% BSA and 1% casein in the AP buffer for 1 h at room temperature, and then incubated for 15 min in a solution of 1 µg/ mL of streptavidin-AP conjugate prepared on the same buffer. Membranes were then rinsed 4 times with the AP buffer and placed, first, in the AP buffer with pH ) 9.5 and then in the standard solution of the AP substrate. (ii) ESD on Glass. A solution of λ DNA was prepared as described above but without glycerol added. After ESD (+(4.05.3) kV; 20-40 nA; humidity, 10-30%) on plasma cleaned glass prepared as described in ref 27, DNA dots were UV cross-linked for 15 min, washed for 15 min in TBS buffer (0.14 M NaCl/2.7 mM KCl/25 mM TRIS/HCl, pH ) 7.4) at room temperature, and then hybridized for 14 h at 62 °C in 4 × SSC solution containing 1% SDS, 10% dextran sulfate, 100 µg/mL of sonicated salmon DNA, and 100 ng/mL of biotinilated λ-DNA probe. The glass was then washed twice for 5 min in 2 × SSC containing 0.2% SDS, washed once for 5 min in 0.2 × SSC, and blocked for 30 min at room temperature in 2% BSA/1% casein in the AP buffer, pH ) 7.5. After washing with the AP buffer, the substrate was incubated for 25 min in a solution of the AP-labeled streptavidin (25 µg/mL) with shaking, washed in the AP buffer, and incubated in the AP substrate as described above. RESULTS AND DISCUSSION The main challenge of the present work was to develop a new technology and to test its feasibility in a variety of conditions. For this purpose, different substances were ES-deposited using masks and substrates made of different materials. The results of such testing are presented below together with a discussion of the essential characteristics of ESD and retention of functional properties of the ES-deposited biological molecules. Different ESD Methods. Of the above suggested methods to control the ESD, only the dielectric mask with an array of holes (27) Morozov, V. N.; Morozova, T. Ya.; Hiort, C.; Schwartz, D. C. J. Microsc. (Oxford) 1996, 183, 205-214.
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Table 1. Efficiency of ESD through Different Dielectric Masks mask material unknowne Teflon Teflonf mica nylong
holes diam open ESD on ESD on efficiency µm area,a % substr,b % mask,c % vs shadowd 370 1540 990 75 25
4.1 10.9 4.9 0.18 25
27 65 51 15 55
57 14 2 65 7
6.6 6.0 10 83 2.2
a Fraction of the mask surface occupied by holes. b Fraction of the electrosprayed material found on substrate. c Fraction of the electrosprayed material found on mask. d Efficiency of dielectric mask related to that of conducting (shadow) mask with the same holes. It is calculated as a ratio of the electrosprayed material found on the substrate after the ESD through the dielectric mask (column 4) to the quantity of the material expected to be found on the substrate in case of even deposition on the conducting mask and on open substrate areas (column 3). e Mask presented in Figure 2B. f Mask through which the array shown in Figure 5A was deposited. g Mask shown in Figure 2A.
and the ESD on an illuminated photoconductive layer have been tested here. As illustrated in Figs. 2, 4, and 5, both methods allow production of arrays of spots of different substances. Comparison of the deposition patterns presented in Figs. 2C, 2D, and 2F shows that photo-ESD is characterized by a larger background. Deposition outside the illuminated areas may be caused both by stray light and by a notable dark conductivity of the photoconductive layer. Proper choice of photoconductive materials and use of a photomask should considerably reduce the background. Such a technically simple optical method can be effectively used to control spatial deposition in cases when cross-contamination of dots is not crucial. The method of ESD through a dielectric mask with holes has an obvious advantage over the photo-ESD method since it completely protects unexposed parts of the substrate from the deposition. Therefore, cross-contamination of dots is entirely avoided. That is why all further results and discussion will concern only the ESD through dielectric masks. Efficiency of the ESD. Deposition through a dielectric mask is highly economical. After the first portion of the charged products is adsorbed on the mask, its surface becomes charged with the same sign and prevents further landing of the products, directing them into the holes. Such a concentrating effect inherent in the dielectric mask results in a much higher efficiency of ESD through such mask as compared with that through a conducting (shadow) one19 where ESD occurs evenly onto both substrate and mask. Therefore, theoretical efficiency of the shadow mask is equal to the fraction of mask surface occupied by holes. As seen from Table 1 efficiency of ESD through dielectric masks used in our experiments varied between 15 and 65%, considerably exceeding the theoretical efficiency of the shadow masks of similar design. Masks with a small open area have an especially large gain in efficiency (83 times for the mica mask with 0.18% open area). Materials for Masks and Substrates. Having in mind the development of a technique feasible for different applications, we tested substrates and masks made of different materials. Some of the masks and results of the ESD on different substrates are shown in Figs. 2, 4, and 5. It was found that, in addition to metals (Al, Au) and semiconductors (SnO2), some dielectrics (plasmacleaned glass, freshly cleaved mica, paper, and membranes) are
suitable for making substrates. In the latter case, discharge of the incoming ES products presumably occurs as a result of a slight ionic surface and/or bulk conductivity in these dielectrics. Simple estimates show that a film of 100-µm-thick dielectric with a specific bulk conductivity of 10-10 - 10-11 S‚cm-1 (inherent in soda-lime glass, dry paper, cellulose acetate, and other materials28) provides enough conductivity for the effective neutralization of incoming charges at a current of 50 nA per 1000 holes 100 × 100 µm2 each. Increased conductivity of the dielectric substrate at high electric fields24 further reduces the requirements for conductivity of a dielectric substrate. Whatever the mechanism of the conductivity in dielectrics is, the practically important result of this study is that many dielectrics, including membranes routinely used in immunochemical and genetic assays, are suitable for microarray fabrication by the ESD method. Quite different requirements are imposed on the material of the mask. To diminish the loss of the ES products, i.e., to increase the concentrating effect of the mask, the mask material should have low bulk and surface conductivity in a high electric field, low mobility, and strong trapping of injected charge carriers. We have found that in this respect mica is a better material than plastics are. Masks made of mica were less contaminated with the ES products than the masks made of plastics (Teflon, polyethylene, and polystyrene) which presumably reflects a higher mobility of the injected charges in the plastics.24 It is practically important that fabrication of micron-sized dots does not require similarly small holes in a mask. As it was mentioned in early publications on the ESD fabrication of radioactive films,11-14 the size of the ES deposited spots can be notably smaller than that of the hole in the dielectric mask. This effect may be called the lens effect. Its physical mechanism still remains unclear. The theory of vacuum electrostatic lenses is not applicable to the lens effect under the normal pressure since the former is principally based on interplay of the electrostatic and inertial forces, whereas the friction forces dominate over the inertial ones in the ambient air. The electrostatic lens effect is readily observed with arrays of holes. It reduces technical difficulties in fabrication of masks for micromatrixes. Thus, a simple fabric with holes of 25 µm, shown in Figure 2A, allows a fabrication of dots as small as 2-6 µm, as can be seen in Figures 2C, 2D, and 2E. Hence, ESD has the potential to greatly miniaturize biochemical assay elements: a matrix containing as many as 1 × 105 dots can be, in principle, deposited on an area of 1 × 1 mm2. Of course, manufacturing of such tiny matrixes requires special rigid, flat masks and substrates, a precise micropositioner, and a dust-free environment. However, matrixes with elements exceeding 50 µm in size can be readily fabricated in a conventional laboratory. Two examples of such multicomponent matrixes are presented in Figure 5. Uniformity of the ESD. An important requirement to the ESD technique consists in obtaining an even distribution of the electrosprayed material among all the dots. There are two factors responsible for the variation in the dot size. First is a variation in the distance between the substrate surface and the mask. Fabrication of completely flat masks and substrates or introduction of spacers between flexible masks and rigid substrates can solve (28) Tables of Physical and Chemical Constants; Longman Group Limited: London, 1993; p 107.
Figure 3. Radial distribution of the surface density in the ES deposit. The number of electrosprayed glycerol microdroplets per unit area was counted under an optical microscope as described in ref 22. ESD was performed in the presence of a perforated cylindrical dielectric shield 40 mm in diameter. Open and closed circles for deposition from a capillary placed at a heights of 30 and 43 mm over the substrate, respectively.
Figure 4. Functional activity of biological molecules in the ESdeposited arrays. Arrays of the peroxidase spots (A) ES deposited onto a nitrocellulose membrane and the AP spots (B) deposited onto Immobilon-P membrane through holes in the plastic mask shown in Figure 2B. The spots are visualized by colored insoluble products of the catalytic reactions. (C) Array of the λ DNA dots ES deposited on a Gene Screen membrane hybridized with the complimentary biotinilated λ DNA probes, developed by reaction with streptavidin-AP conjugate and colored with the insoluble product of the AP reaction. (D) Similar dots deposited onto a plasma-cleaned glass. The distance between spots is 1 mm in A and B, 75 µm in C and D.
this problem. The second factor originates from a bell-shaped distribution of the deposit density with a maximum under the capillary tip.29 Two systems were tested as means of equalizing the substance distribution over the whole substrate area. In one of them, the mask and the substrate were combined in one unit, which was placed on a rotating table. The capillary tip periodically oscillated to spend equal time over all areas of the rotating substrate as described in ref 12. As shown in Figure 5A, a fair uniformity is achieved with this method. (29) Jones, A. E. In Essays on Formal Aspects of Electromagnetic Theory. Lakhtakia, A., Ed.; World Scientific Publ. Co.: New York, 1993; pp 228-267.
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Figure 5. Multicomponent matrixes of synthetic and biological substances. (A) Libraries of 36 different synthetic dyes ES deposited onto an Immobilon-P membrane using a Teflon mask with round holes 0.99 mm in diameter spaced 10 mm apart. (B) Matrixes of 4 antigens (BSA, HSA, HHb, and OA) ES deposited through the mask shown in Figure 2B onto the Immobilon-P membrane surface and developed with the dot immunobinding technique as described in the Methods section.
Relative motion of the capillary and substrate is an effective but complex way to equalize the deposition density. In the another system, a perforated cylinder shield was placed axially over the deposition area as shown schematically in Figures 1A and 1B. The shield was made of a woven polymer tissue similar to that shown in Figure 2A. We found that introduction of the shield results in a remarkably even distribution of the deposit (see Figure 3) provided the following conditions are satisfied: (i) the shield’s diameter is smaller than the diameter of the deposit in the absence of the shield, (ii) the diameter of the target electrode exceeds that of the shield, and (iii) the size of each hole in the shield is smaller than 0.5 mm (otherwise the charged products penetrate through the shield). Though the mechanism of this density redistribution is not clear at present, we assume that the electrostatic confinement of the “cloud” of charged products by the charged shield is responsible for the effect. Retention of Functional Activity of Biological Molecules upon the ESD. Some biological molecules, such as proteins, are fragile and can rapidly lose their functional activity upon adsorption, drying, and other manipulations. To these factors, the ESD potentially adds others, such as changes in pH as result of 3116 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999
electrochemical reaction on electrode in the capillary, direct electrochemical reaction on the electrode, reaction with chemically active products of corona discharge and the impact of the charged particles with substrate surface. Fortunately, proper choice of the ESD conditions allows the elimination of the damage and the obtainal of nearly 100% preservation of specific enzyme activity, as it has been demonstrated recently.23 In short, ESD has to be performed at low voltage and current (
