Generation of biologically active nano-aerosol by an electrospray-neutralization method

Journal of Aerosol Science 42 (2011) 341–354

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Generation of biologically active nano-aerosol by an electrospray-neutralization method Victor N. Morozov a,b,n a b

National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushchino, Moscow Region 142290, Russia

a r t i c l e i n f o

abstract

Article history: Received 21 October 2010 Received in revised form 16 February 2011 Accepted 16 February 2011 Available online 22 February 2011

A simple method for manufacturing biological nano-aerosols is described. It is based on gas-phase neutralization of a cloud of highly charged electrospray-generated particles or macromolecular ions with a cloud of oppositely charged electrospray products, e.g., ions of a volatile solvent. It was demonstrated that the electrosprayed products became neutralized within a few seconds, forming a stable nano-aerosol composed of single polymer molecules, nanofibers, or nano-clusters of different sizes, depending on polymer concentration, solvent, humidity, and other factors. It was also demonstrated that enzymes aerosolized by this mild technique retain their specific activity, which opens a variety of new applications for this technology. & 2011 Elsevier Ltd. All rights reserved.

Keywords: Electrospray Nano-aerosol Neutralization Enzyme

1. Introduction Generation of aerosols by electrospraying (ES) of solutions was first described by Zeleny (1917). Later Dole et al. (1968), Fenn (2002) and his coworkers (Yamashita & Fenn, 1984; Wong, Meng, & Fenn, 1988), as well as a group of Russian researchers (Alexandrov et al., 1984; Kozenkov & Fuks, 1976; Zolotoi, Karpov, & Skurat, 1988) demonstrated the feasibility of electrospraying as a soft ionization technique for mass spectrometry. These publications in electrospray ionization (ESI) methods revived interest in the electrospray atomization technique as a means of generating mono-disperse microdroplets, manufacturing solid nano-clusters, nanofibers and nanotubes (see excellent reviews of Corn & Esman, 1976; Greiner & Wendorf, 2007; Kebarle, 2000; Li & Xia, 2004; Salata, 2005; Tang & Gomez, 1995). Among the variety of aerosol generation techniques, ES atomization stands out as the simplest and most energy-efficient technique capable of producing the smallest particles. However, highly charged ES-generated aerosol is intrinsically unstable: the cloud rapidly expands due to space charge repulsion, and the charged aerosol particles quickly settle on the walls. To increase aerosol stability, partial or complete neutralization is conventionally performed in contact with air ionized by a radioactive isotope (Basak, Chen, & Biswas, 2007; Bacher et al., 2001; Scalf, Westphall, Krause, Kaufman, & Smith, 1999; Welle & Jacobsa, 2005) or by a corona discharge (De La Mora, Navascues, Fernandez, & Rosell-Llompart, 1990; Ijesebaert, Geerse, Marijnisseen, Lammers, & Zanen, 2001; Kozenkov & Fuks, 1976). In both of these neutralization techniques, aerosol particles are exposed to highly reactive ionization products: radicals, hot molecules, ozone and oxygen atoms; all are destructive for polymer and biological macromolecules as well as for living cells, spores, or viruses. Using atomic force microscopy, the author has observed fragmentation of electrospray-deposited polymer molecules upon their neutralization on a mica surface with

n Correspondence address. Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushchino, Moscow region 142290, Russia. Tel.: + 7 496 773 0623. E-mail address: [email protected]

0021-8502/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaerosci.2011.02.008

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corona-generated counter-ions (Morozov, 2010). Oxidation and ion-attachment reactions have also been documented upon charge reduction in ESI-MS with both radioactive and corona neutralizers (Frey, Lin, Westphall, & Smith, 2005). Therefore, neutralization with gaseous counter-ions generated by a soft ES technique seems an attractive alternative to radioactive and corona neutralizers. The author is aware of only two publications which describe mixing of oppositely charged electrospray-generated clouds. Camelot, Marijnissen, and Scarlett (1999) reported a rapid mixing of two reagents by coalescence of oppositely charged microdroplets generated by ES. Solid products precipitated in the coalesced droplets and turned into monodisperse particles after solvent evaporation. Almekinders and Jones (1999) described the formation of a zero-charged aerosol by mixing positively and negatively charged microdroplets produced by electrohydrodynamic atomization. Though the authors claimed that the oppositely charged droplets did not coalesce in their experiments, no evidence has been provided to support such a conclusion. In our recent papers, we demonstrated that gas-phase neutralization of electrospun polymer nanofibers with ES-generated ethanol counter-ions enabled the manufacture of free nanomats (Morozov & Vsevolodov, 2007). Such nanomats could be used as highly effective filters for collection and detection of bio-aerosols (Vetcher, Gearheart, & Morozov, 2008). One may generalize this approach still further by combining a variety of electrospray-generated oppositely charged products such as micro- and nano-droplets, small solvent ions, macromolecular ions, nano-clusters of non-volatile substances, and fibers. The presence of the charges enables the outcome of ‘‘mixing’’ the ES-generated products to be controlled by inhibiting the collisions between similarly charged species and accelerating those between the oppositely charged electrospray products. Here the process of electrospray neutralization (ESN) is studied in more detail to reveal major factors controlling the phenomena of mutual neutralization of ES-generated nano-particles and to characterize how protein molecules survive this type of aerosolizing procedure. 2. Materials and methods 2.1. Materials Alkaline phosphatase from bovine intestinal mucosa (PA), ovalbumin (OVA), bovine serum albumin (BSA), FITC-labeled BSA (FITC-BSA), gelatin from bovine skin (type A), polyvinylpyrrolidone (PVP, Mw = 360,000), polyvinyl alcohol (PVA with Mw = 31–50 kDa), polyethyleneimine (PEI, Mw = 750,000), glutaraldehyde (GA, 25% solution), TRIS/HCl, NaCl, NaN3, Tween20, ethylenediaminetetraacetic acid (EDTA), urea, absolute ethanol, NN-dimethylformamide (DMFA) and para-nitrophenol (pNPP tablets) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Urease (from Canavalia ensiformis, or Jack beans) was obtained from Serva (Heidelberg, Germany). All proteins were thoroughly dialyzed against milli-Q water before use. Conductivity of dialyzed 1% BSA solution was 30–40 mS/cm. 2.2. Design of nano-aerosol generators Three types of nano-aerosol generator were designed during this study. The simplest aerosol generator, schematically illustrated in Fig. 1A, consists of a nearly spherical plastic chamber (1 liter by volume) which has two holes for the introduction of electrospray capillaries and two hoses for pumping air in and out. One capillary is filled with a solution or suspension to be transformed into aerosol. The second capillary is filled with a volatile solvent. The solution in each capillary is connected to a high-voltage power supply with a platinum wire electrode; the design is described in our recent papers (Morozov & Morozova, 1999a, 1999b). In addition to the elements just mentioned, the prototype contains three other features: (i) a fan, (ii) a temperature/humidity sensor (not shown in the schematic of Fig. 1A), and (iii) a port for introduction of a microscopic slide with a piece of mica attached. The latter was used to collect aerosol particles and fibers for further analysis with optical and atomic force microscopy. The fan ensured even distribution of the aerosol throughout the chamber. The relative humidity was controlled with a commercial digital hygrometer and adjusted by introducing air dried over silica gel or humidified air into the chamber (see more details in the following section). The ESN device presented in Figs 1A and 2A did not permit characterization of the initial size distribution of nanoaerosol particles generated in ESN because of the relatively large volume of the chamber which led to substantial aggregation of nano-particles before their deposition on mica. To solve this problem, a smaller ESN chamber was designed as illustrated in Figs. 1B and 2B. With a volume of only 40 mL, it allowed for quick delivery of generated nano-aerosol into a Scanning Mobility Particle Sizer (SMPS, from TSI Inc., Shoreview, MN). The distance between the capillary tips was adjustable from 10 to 50 mm. Two glass windows (see image of the cell in Fig. 2B) glued into the cell walls were used to illuminate the ES torch with a laser beam and to observe the ES torch. The cell was connected to a bag filled with filtered air as shown schematically in Fig. 3. Another piece of conductive elastic tubing, 5 mm ID and 10–20 cm long, connected the cell to the SMPS device. Thus, at a flow rate of 0.6 L/min, aerosol products reached the classifier (catalog #3080, from TSI Inc.) in  0.4 s. Because the total cell volume of 40 mL is replaced at that flow rate in 4 s, we will take the latter number as an estimate of the time needed for the ESN products to reach the SMPS device. The third apparatus was designed to reveal the spatial distribution of the aerosol particles in the space between the ES capillary tips. Its schematic is presented in Fig. 1C, and its image in Fig. 2C. It was made of a plastic cylinder (1), 152 mm in

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Fan Positive cloud

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Air in

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Port + Air in F 5

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Fig. 1. Schematics of three chambers used for generation of nano-aerosol particles by the electrospray-neutralization (ESN) method. (A) Large-volume chamber. (B) Small-volume flow-through chamber. (C) Schematic of a laminar flow-through chamber used to study spatial distribution of concentration and size of ESN-generated nano-aerosol.

Fig. 2. Images of nano-aerosol generators used in this study. (A) First prototype of the apparatus schematically illustrated in Fig. 1A. (B) A flow-through generator with a small volume mixing chamber (schematic in Fig. 1B). (C) A flow chamber used in the experiments on spatial distribution of aerosol as schematized in Fig. 1C. The foam plate covering the cell has been removed.

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3 6

Compressed air 1

7

2 4

5

5 1

Fig. 3. Schematic design of an aerosol generator employing a flow-through ESN chamber (Figs. 1B and 2B).

diameter, 133 mm high, with a plastic plate (2) glued to the bottom. A fan (3) attached to the bottom pumped air through a wide (70 mm in diameter) hole in the bottom plate. A porous tissue (4) covered the top of the cell, protecting it from occasional air motions. The chamber had two portholes (5), 120 mm from the bottom, used for introduction of capillary holders. Another porthole (7), placed 20 mm above the bottom, was used to introduce a metal tube (6) (4 mm ID) for aspiration of aerosol at different positions with respect to the capillary tips. The vertical air flow created by the fan was measured with an air flow meter (Testo 415, GmbH & Co., Germany). The fan created a downward air velocity of 0.40 70.02 m/s, which at the level of the capillaries was uniform within a radius of 3.5 cm around the axial line of the cylinder jar. As aspiration of air into the tube (6) at a volume rate of 10 mL/s was much lower than the overall volumetric downward flow rate exceeding 1.5 L/s, we expected that aspiration would not disturb the flow distribution in the chamber significantly. Aerosol particles and neutralizing ions moved downward to the aspiration tube in  0.25 s. A conductive rubber tube (total length 60 cm, 5 mm ID) was used to connect the aspiration tube to the condensation particle counter (CPC) or to the SMPS device. Via this tube, aerosol particles reached the CPC (consuming 5 mL/s) and SMPS (consuming 10 mL/s) in less than 2–3 s. 2.3. Control of air purity and Humidity To control air purity, an experimental set was designed as shown schematically in Fig. 3. Dry air flowed through a valve (1) and a HEPA capsule filter (2) (TSI, Inc., catalog # 1602051, retains 99.97% of particles 4 300 nm) into a plastic bag (3) equipped with a fan (4) and a hygrometer (not shown in Fig. 3). Air from the bag (3) was pumped through the smallvolume ESN cell (6) (see Figs. 1B and 2B) into the SMPS device after being mixed with a metered flow of dried purified air. Flow meters (5) served to control flow rates. The cell was connected either to the CPC or to the SMPS device by conductive elastic tubing, 5 mm ID and  20 cm long. To study the effects of humidity on aerosol size distribution, the plastic bag (3) was filled with filtered air of certain humidity. To avoid problems with dew formation in the SMPS, the air flow at the output of the ESN chamber was mixed with an equal flow of purified air dried over silica gel. Thus, when the air in the ESN chamber was 100%, the humidity of the air mixture entering the SMPS was 50%. Hazards. The high-voltage supply used in the device requires some caution. It is recommended to connect the output via a resistance of 50–100 MO to limit the current. 2.3.1. Aerosol analyzer Aerosol size spectra were obtained with a scanning mobility particle sizer from TSI Inc. (Shoreview, MN) consisting of the model 3080 and 3085 differential mobility analyzers (DMA) and the model 3786 condensation particle counter (CPC) under the following typical conditions: impactor of 0.071 cm, DMA flow rate of 6 L/min, and CPC inlet flow rate of 0.3 L/min. Corrections for multiple charges and diffusion losses were introduced as recommended by the manufacturer. In studies of humidity effects, the sensor of a digital hygrometer was introduced into the bag (3) schematized in Fig. 3, and the air inside the bag was dried by passing the filtered air through a silica gel column or humidified by introducing strips of Whatman paper wetted with a metered amount of water. 2.4. AFM imaging A slightly modified Nano-RMTM atomic force microscope (Pacific Nanotechnology, Santa Clara, CA) was used for AFM measurements. Tygon tubing was attached close to the scanning head in order to direct a weak jet of air dried over silica gel onto the scanning cantilever and the substrate surface. Keeping the environment dry prevents formation of a water bridge between the tip and the mica surface and eliminates the effects of humidity. A tapping mode with a resonance frequency of 300–350 kHz was used in all scanning experiments. Tip quality was routinely controlled by scanning samples of electrospun PVP solutions which presented a variety of fibers from sub-nanometer height to 500 nm (Morozov and Morozova, 1998). Cantilevers which did not reveal fibers 1 nm high were discarded.

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In a typical experiment, mica was pre-treated in a 1% solution of poly(ethyleneimine) to enhance binding of protein nanostructures to the surface. Protein aerosol generated in the chamber (Figs. 1A and 2A) was allowed to settle on the mica surface for a few minutes. Then the sample was exposed for 2–5 min to glutaraldehyde (GA) vapor to prevent molecules and clusters from displacement by the cantilever tip upon scanning. To decrease water activity in the 25% commercial GA solution, dry NaCl powder was added to the solution in large excess. The relative humidity over this NaCl slurry was reduced to  77%. The procedure of GA fixation at such moderate humidity prevented protein molecules and clusters from changing shape as a result of interacting with the solid surface. Gelatin nano-aerosol particles and fibers were collected for 5 min on freshly cleaved mica when dialyzed gelatin solution was sprayed against pure ethanol. 2.5. Retention of enzyme specific activity in protein aerosol Commercial alkaline phosphatase (AP) was dissolved in water and dialyzed against water overnight at 4 1C to give an electric conductivity of s = 10–13 mS cm 1. The protein concentration was determined using an extinction coefficient of 280 nm according to Landt, Boltz, and Butler (1978). The AP solution, with a protein concentration of 1.470.1 mg/mL, was mixed with an equal volume of dialyzed FITC-labeled BSA solution with a protein concentration of 3.2 mg/mL. The latter was measured using a quartz crystal microbalance (Morozov & Morozova, 1999a, 1999b). Microliter aliquots of this mixture were added to 2 mL of a blocking solution (2% PVA in 20 mM TRIS/HCl buffer, pH= 7.5, containing 0.15 M NaCl, 0.05% Tween-20 and 0.02% NaN3), and the fluorescence intensity was measured using the Picofluor Handheld Dual Channel Fluorometer (Turner BioSystems, Sunnyvale, CA). A graph of the fluorescence vs. added volume of the AP/BSA-FITC mixture was used as a calibration curve. The mixture of AP and FITC-BSA (2.570.1 mL) was placed into a glass capillary and the capillary was inserted into the aerosol generator chamber shown in Figs. 1A and 2A. A second capillary was filled with  10 mL of absolute ethanol. A positive pole of a high-voltage power supply (6–8 kV) was connected to the platinum wire inside the first capillary, while the negative pole was connected to a similar electrode in the second capillary, resulting in a steady current of 50 720 nA. The distance between the capillary tips (30–50 mm in OD) was set to 60–80 mm. To accelerate the process, a pressure of 18–20 cm of water was applied to the capillary with the AP/BSA-FITC mixture. The initial air humidity in the chamber was 22%. The entire volume of the mixture was aerosolized in 5–6 min, increasing the humidity to 33%. The chamber was kept closed for another 10 min to let the aerosol settle on the walls. The chamber was then opened in a hood and 2.5 mL of the blocking solution was placed inside the chamber (thereby making a 1000-fold dilution of the sprayed protein mixture). The chamber was rotated so that the whole surface was brought into contact with the buffer. Typically 1.5–1.7 mL of the buffer was collected for further analysis. The fluorescence of the collected solution was measured and used to determine the efficiency of aerosol collection. To compare specific AP activity before and after electrospraying, 10 mL probes of the initial AP/BSA-FITC mixture diluted 1:1000 with the blocking solution and 10 mL probes of the solution collected after the chamber washing were each added to 1 mL of commercial pNPP substrate, and the rates of substrate hydrolysis were measured by monitoring changes in the optical density at 405 nm. Each rate was then divided by the fluorescence intensity of the respective probe to account for the enzyme loss in the collected sample. The ratio of the normalized enzymatic activity in the collected sample and the normalized initial activity of this enzyme was used as a measure of retention of specific enzyme activity in the aerosolized AP. 2.6. Comparison of enzyme activity retained in protein aerosol neutralized by counter-ions generated by electrospraying and by corona discharge Urease from Jack beans was thoroughly dialyzed against a large excess of 0.2 mM EDTA solution at 4oC. The urease solution (3.2 mg/mL, conductivity, s =50 mS cm 1) was electrosprayed within the chamber presented in Figs. 1A and 2A, first against ethanol then against a corona source: sharpened Pt wire, 0.2 mm in diameter, 4 mm long. The distance between the capillary tips was set to 100 mm. The corona ionizer was placed in the same position as the capillary tip filled with ethanol. One chamber hose was connected to a water-soluble PVP nano-filter described in our previous paper (Vetcher et al., 2008); the filter was further attached to a membrane vacuum pump with a pumping rate of 4 L/min. The air pumped from the chamber was replaced with fresh air filtered through a Whatman HEPA-VENT capsule (Cole-Parmer Instrument Company, Vernon Hills, IL) connected to the second hose in the chamber. After electrospraying 2–4 mL of the urease solution over 1870.5 min at a steady current of 200 720 nA, the PVP filter was dissolved in 20 mL of water. This solution was added to 0.5 mL of a substrate solution: 0.15 M urea dissolved in 5 mM phosphate buffer, pH= 7.6. Changes in pH due to decomposition of urea by added urease were registered by a chart recorder and used to calculate the enzyme reaction rate. The calculated rate was compared to the urease activity in a similar volume of the initial urease solution used for spraying. 3. Results and discussion The ESN process starts at a voltage higher than that in electrospray deposition (ESD) on a conductive plate placed at a distance equal to the distance between the capillary tips. This is explained by the two large potential drops (in the vicinity

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I. Standard electrospray

II. Electrospray with neutralization

Dry residue of mother and daughter drops

Dry neutral residue of mother drop

Fig. 4. Major difference in the fate of a droplet produced by ES in a standard ES (upper scenario) and in an ES with neutralization (lower scenario). A poly-disperse nano-aerosol is produced in a dry atmosphere in the first procedure. The second procedure yields a mono-disperse aerosol.

of each capillary tip) in the ESN, as opposed to one in the ESD. Once the critical voltage is reached, stable torches as well as Taylor cones become visible on both capillary tips, and a stable current goes through the capillaries, provided the chamber is not touched by a hand or any conductive object. One may thus conclude that the electrospraying process in the ESN is entirely controlled by a local electric field at each tip and that the electrospraying proceeds exactly as in the ESD and ESIMS processes, following the same dependence of the size and charge of the generated microdroplets upon conductivity, surface tension of liquid, and flow rate (Corn & Esman, 1976; Greiner & Wendorf, 2007; Kebarle, 2000; Tang & Gomez, 1995). Electrospray neutralization as described here results in an exact zero net charge of the aerosol since positively and negatively charged products are generated at equal rates, thereby producing a bipolar ‘‘plasma’’ which undergoes neutralization in parallel with the decomposition of charged microdroplets. It is well known that a jet of electrosprayed mother droplets emitted from the capillary tip is transformed into a cloud of highly charged dry residues of progeny nanodroplets as the result of a series of electrostatic fissions following solvent evaporation from the mother droplets (Kebarle, 2000), as shown schematically in the upper part of Fig. 4. In electrospray deposition, charged electrospray products are neutralized by giving or accepting electrons from a conductive substrate. Thus, in electrospray deposition, the neutralization process is completely separated from the atomization process. In ESN, these two processes occur simultaneously in the gas phase and can affect each other. One may expect, for example, that larger aerosol particles will be produced in the ESN, if neutralizing counter-ions reach the charged aerosol microdroplets before the latter experience the full set of drying-decaying cycles. In the ultimate case, when neutralization of mother droplets happens before the first electrostatic decay, as illustrated in the lower part of Fig. 4, all the non-volatile content of the mother droplet will end up in a single dry residue particle. One may expect from such scenarios that any conditions which reduce the speed of solvent evaporation from the mother droplet will favor the formation of larger particles. Because of this competition between neutralization and electrostatic decay of droplets, the size distribution of the generated particles should strongly depend on the rate of solvent evaporation from the mother droplet and on the mobility of the counterions. Since at higher humidity ES-generated microdroplets evaporate more slowly, they have a good chance of becoming neutralized before reaching the Raleigh limit. At low humidity, the droplet has a chance to disintegrate before neutralization happens, so generation of much smaller dry residue particles from the daughter nano-droplets is expected. These predictions have been well-supported by the experimental data presented below.

3.1. Dependence of nano-aerosol size on air humidity and solvent evaporation rate. Profound effects of humidity on nano-aerosol generation by ESN were readily seen in both AFM images and in spectra obtained with the aerosol sizer. Data presented in Fig. 5 demonstrate that ESN at a moderate (A= 75%) and high (A= 98%) humidity resulted in notably different height distributions in the PVA nano-aerosol particles: while all PVA particles generated at moderate humidity have heights lower than 30 nm, many particles with larger heights were observed on the mica surface when ESN was performed at high humidity. This difference cannot be explained by a difference in the flattening of the PVA particles in contact with the mica surface at different humidities, since at high humidity the hydrophilic PVA particles should be more prone to deformation and should acquire a flatter shape in contact with the surface than at moderate humidity. We conclude that larger aerosol particles are generated at higher humidity in good accordance with the scenario schematized in Fig. 4. The ESN process with the same PVA dissolved in NN-dimethylformamide (DMFA) resulted in notably larger particles as compared with those generated at the same low humidity from an aqueous solution of PVA of similar concentration. One can see from the histograms of PVA particles presented in Fig. 6 that fewer small particles and more large particles were generated from the solution in DMFA than in water under otherwise identical conditions. This difference can be readily

V.N. Morozov / Journal of Aerosol Science 42 (2011) 341–354

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45 40

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Fig. 5. Comparison of the distribution of aerosol particles generated from a 1% PVA solution in water at high relative humidity (black bars, 98%) and at moderate humidity (white bars, 75 75%). The aerosol was generated in the chamber depicted in Fig. 1A. Absolute ethanol was used to generate negative counter-ions.

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explained by the notably higher boiling point of DMFA (153 1C) and, hence, the slower evaporation rate of DMFA from the mother droplets as compared to that of water. The slower evaporation rate increased the probability of neutralization of the mother droplets before electrostatic decay in good accordance with the ESN scenario shown in Fig. 4. Of course, part of the difference may originate from the difference in the electrospraying process for these two solvents, which have different densities, viscosities, and surface tensions—factors known to affect the radius of electrosprayed mother droplets (Tang & Gomez, 1995; Kebarle, 2000). In using AFM to characterize aerosol size, one should take into account that the aerosol spectra are distorted in at least three ways: (i) due to changes in the particle shape in contact with the substrate surface, as discussed above, (ii) due to aerosol aggregation before landing, and (iii) due to differences in the diffusion-controlled deposition rates for particles of different sizes. While the second factor is expected to decrease the fraction of observed small particles, the third one leads to underrepresentation of larger particles in the AFM images. These limitations of the AFM technique do not affect the qualitative conclusions drawn above regarding the effects of humidity in ESN. The conclusions were further supported by spectra obtained with the SMPS device. The humidity dependence of aerosol spectra was also evaluated by the SMPS device using 1% solutions of both sucrose and dialyzed BSA. The results for the sucrose aerosol are presented in a series of panels in Fig. 7. In accordance with our expectations, the spectrum of the nano-aerosol obtained by ESN at a very low humidity (panel A in Fig. 7) is drastically different from those obtained at moderate and high humidities. The presence of a large fraction of small nano-particles with a broad distribution of sizes indicates that at low humidity, the mother droplets had a chance to decompose and produce numerous small progeny nano-droplets. The spectra of the ESN-produced aerosol at moderate and high humidity (panels B and C in Fig. 7) were quite different: far fewer small particles were generated and an almost mono-disperse nano-aerosol with a major peak at 20 nm was produced. The latter size still cannot be attributed to the dry residue of the entire mother droplet, since the dry residue with a diameter of 20 nm would require evaporation of a 1% sucrose microdroplet with a diameter of 130 nm—an order of magnitude smaller than the average size estimated for the mother droplets (Morozov and Morozova, 1998). Even in saturated water vapor (when several exhales were made into the bag (3) to ensure vapor saturation, so that condensation was seen on its inner surface), the size of the sucrose clusters increased by

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Fig. 7. Spectra of aerosol manufactured from 1% sucrose solution in water by the ESN process with EtOH neutralization at low humidity (panel A for air dried over silica gel), at moderate humidity (panel B, A = 65–70%) and at high humidity (panel C, A = 94–97%; panel D, 100% humidity). Other parameters: current 18–20 nA, distance between capillary tips 50 mm. Corrections for multiple charges and diffusion losses were introduced. The number concentration, dN, measured by the SMPS spectrometer is the concentration of particles in a given size channel.

only  30%, as seen in Fig. 7C. This increase may be attributed to incomplete dehydration of sucrose nano-clusters at 50% humidity inside the SMPC. One potential explanation for the presence of small nano-particles at high humidity is that mixing EtOH and sucrose droplets results in decreased surface tension, which facilitates the decomposition of EtOH-water droplets even at 100% humidity. This mechanism was ruled out in special experiments in which EtOH neutralization was replaced with neutralization by corona products from the sharp platinum needle described above. The humidity dependence of aerosol sizes was similar to that observed in neutralization with EtOH counter-ions. One may thus conclude that the mother droplets undergo some Coulomb fission even at 100% humidity. It may happen near the Taylor cone as a result of destabilization of the charged droplets in a highly uneven electrostatic field; such an idea has been discussed by Siu, Guevremont, Le Blanc, O’Brien, and Berman (1993) and by Krasnov and Shevchenko (1995).

3.2. Dependence of nanoparticle size on pressure (flow rate of solution) Pressure is another factor capable of changing the spectrum of a nano-aerosol generated by the ESN process. Morozov (2010) mentioned that using a syringe pump to feed the electrospray capillary at a fixed flow rate has the drawback of producing occasional droplets (spills). To avoid these, we controlled the flow rate of solutions through the capillary (in the range of 0.2–0.8 mL/min), not by a pump but by applying a hydrostatic pressure, which increased the flow rate proportionally to the applied pressure and which provided a stable cone-jet electrospray at combinations of flow rate and current different from those at a constant pump-controlled flow rate (Morozov, 2010). A typical spectrum of a nano-aerosol generated with a pressure of 5000 Pa applied to the solution in the capillary is presented in Fig. 8. It contains two peaks: at 30 nm and at 150 nm. Though the concentration of the former is  10 times greater than that of the large particles (compare Figs. 8C and B), they accounted for only  10% of the BSA content in the total aerosol, as is evident from comparing the areas under the first and second peaks in the spectra presented in Fig. 8A. Applying a pressure of 5000 Pa to the protein solution in the capillary resulted in a two-fold increase in the size of smaller nano-clusters (peak at 12–30 nm) of BSA and a ten-fold increase in the rate of their generation. The larger fraction of nano-particles (with a mean diameter ranging from 100 nm at low humidity to 150 nm at high humidity) exhibited fewer changes in the diameter, but the rate of their generation increased  10-fold when a pressure of 5000 Pa was applied. Humidity does not notably affect the pressure dependence of the nano-aerosol diameter and the rate of its generation as one can see from comparison of Fig. 8B and D. Of course, the numbers presented in Fig. 8 will vary with the diameter of tip and the length of the extended thin part of the capillary. The effect of applied pressure just described is similar to the way pressure affects the formation of charged droplets: pressure applied to the solution in the capillary makes it spray more rapidly, resulting in a larger number of microdroplets with larger sizes being generated in the ESN process (Fernandez de la Mora, 2007). A practically important result of studying the effects of pressure consists of the ability to greatly vary the rate of nano-aerosol production without substantially changing the nano-aerosol spectrum—a phenomenon which could be exploited in nano-aerosol generators for medical purposes to control inhaled doses.

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Fig. 8. Effects of pressure on the size and concentration of aerosol produced from 1% BSA solution by the ESN process. Panel (A) is a typical mass concentration spectrum of aerosol at a pressure of 5400 Pa in air dried over silica gel. Panel (B) illustrates the pressure (in cm of water) dependence of concentration and mean diameter for the left part of the spectrum in panel (A). Panel (C) illustrates the pressure dependence of the right peak in the panel (A). The mean diameter of large nano-particles increases from 807 20 nm at pressure p = 0 cm water to 115 7 15 nm at p= 54 cm water. Panel (D) shows the concentration and mean diameter of the peak of the left part of the spectrum in the panel (A) at humidity of 667 3%. Other parameters: current 20–25 nA, distance between capillary tips 50 mm.

Fig. 9. Spectra of nano-aerosol fabricated from 0.1% solution of dialyzed BSA obtained with the relative air humidity of 32%.

85

Kr neutralizer (A) and without the neutralizer (B). ESN at

3.3. Dependence of nanoparticle size on solute concentration The concentration of non-volatile solute in the electrosprayed solution strongly affects the nano-aerosol spectra. Nano-aerosol produced by ESN from 0.05% PVA solution consisted mostly of nano-particles with average heights of 2.370.6 nm (ESN at 45% humidity in the chamber in Fig. 1A) and rare particles 20–30 nm high. The former height corresponds to approximately one half of the diameter expected for a single PVA molecule with a molecular mass of 31–50 kDa collapsed into a spherical ball. It is worth noting that a similar reduction in the height of collapsed polymer globules has been reported for PVA and polyethylene glycol molecules after ES deposition onto a mica surface (Morozov and Morozova, 1998). We may conclude from such similarity in the deformation of neutral and highly charged globules that it is not electrostatic forces resulting from mirror charges but rather direct interaction with the surface that is responsible for the observed flattening of collapsed hydrophilic polymers which lack a stable internal structure. Aerosol particles from rigid globular proteins behave differently. As seen in Fig. 9A, reducing BSA concentration to 0.1% results in the formation of a characteristic prominent peak at a diameter of 6.3–6.7 nm which may be attributed to nano-aerosol particles comprised of single BSA molecules with the X-ray sizes of the BSA molecule (5.5  5.6  12.0 nm3) according to Carter and Ho (1994). This spectrum corresponds well to the data obtained in the AFM analysis, which showed a notable fraction of particles with average heights of 6.4 72.7 nm when BSA aerosol was generated at 34% humidity from 1% solution. In addition to these particles, larger particles with AFM

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Fig. 10. Fibrous aerosol particles obtained by electrospraying 0.4% solution of dialyzed bovine gelatin A at 20–30% humidity. The process was performed at a voltage difference between the capillaries of 9 kV, and a current of 40–60 nA. EtOH negative counter-ions were used for neutralization.

heights of 57 712 nm were also observed. Particles with heights of 5.7 73.1 nm were observed after electrospraying of diluted solutions of ovalbumin (ESN of 0.1% solution at 75% humidity in the chamber), close to the average size of the ovalbumin molecule 7  4.5  5 nm3 according to X-ray analysis (Stein, Leslie, Finch, & Carrell 1991). Thus, ESN of diluted protein solutions produces a nano-aerosol comprised of single protein molecules as the major ESN product. The close agreement between the AFM height and the X-ray dimensions of the molecules thus deposited indicates that the molecules retain their size, shape, and structure upon landing. We envisage that this mild technique may be used to prepare protein molecules for AFM and scanning tunneling microscopy. Only globular nano-particles were observed in the AFM images after ESN of concentrated solutions of globular proteins, while nanofibers were seen after ESN of concentrated fibrous proteins. As one can see from Fig. 10, electrospraying a fibrillar protein, gelatin, at a relatively high concentration and low humidity (A= 20–30%) resulted in formation of aerosol comprised of nanofibers and nano-loops rather than spherical nano-clusters. Similar fibrillar structures have been observed on the mica surface after direct ES deposition (Morozov & Morozova, 1998). However, in the ESN process it takes much longer (minutes, rather than the milliseconds of ESD) for neutral products to reach the mica substrate by diffusion. It seems remarkable that dry neutral gelatin nanofibers with a diameter of only 2–10 nm as determined by the AFM height were rigid enough to keep their shape in dry air for such a long time, even though such a form is expected to be highly unfavorable thermodynamically due to the large surface that is exposed to air.

3.4. Charging of BSA nano-particles generated in the electrospray-neutralization process How quickly do electrospray-generated macroions and nano-clusters lose their charges? As seen in the spectra presented in Fig. 9A, in addition to the monomolecular aerosol particles, a broadly distributed peak of BSA nano-clusters with an average diameter of 22 nm was also observed. When 85Kr radioisotope neutralizer was removed from the SMPS, the spectrum changed dramatically: the peak of single-molecule aerosol particles at 6.4 nm disappeared, but 22 nm clusters were still readily observable. One may thus conclude that the small protein ions completely lost all their charges within the few seconds it took to transfer the ESN-generated nano-aerosol into the SMPS device. Another explanation for the disappearance of single-molecule nano-aerosol particles involves assuming that these reach the SMPS bearing multiple charges, which makes them too mobile to be detected in the electrostatic classifier. In contrast to the single-molecule particles, large BSA nano-clusters were readily observable without neutralizer, and their concentration was nearly identical to that with the neutralizer, as is seen when comparing the two spectra presented in Fig. 9A and B. Theoretical analysis indicates that 9.2% of the particles with a diameter of 22 nm passing through the radioisotope bipolar neutralizer acquire one positive charge and only 0.02% acquire two positive charges (see the operating manual of Model 3936 SMPS). Taking into account that the SMPS software accounts for this charging ratio, and comparing the 22 nm peak intensities in the two spectra in Fig. 9, we concluded that  10% of the 22-nm BSA nano-particles retained one residual positive charge for at least 0.4–4 s after being generated in the ESN process. The quantitative similarity in the 22 nm peaks obtained with and without neutralizer allow us to think that the final stage of neutralization in ESN provides conditions identical to those in the bipolar neutralization (Scalf et al., 1999; Frey et al., 2005). Since initially not only nano-clusters,

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but also hydrated protons, Na + , and other ions comprise the positive cloud, it is possible that the late stages in ESN process may resemble bipolar neutralization, with both positive and negative high-mobility ions playing the same role as in bipolar charging. Small positive and negative ions move faster than larger charged nano-clusters. Therefore, the latter may arrive to the scene where small ions already have formed a bipolar mixture and where incoming charged nano-clusters may acquire an equilibrium charging state. These hypotheses about the mechanisms of neutralization have to be verified experimentally, a task that goes beyond the scope of this research. 3.5. Spatial distribution of neutralization in the ESN process The neutralization process in a dense gas phase involves a complex aerodynamic interaction of two ionic winds induced by the motion of electrospray products and counter-ions. Where between the two capillary tips do those two ionic winds meet? To answer this question, we studied neutralization under conditions which prevented the mixing of newly formed products with previously formed products. This was achieved by performing neutralization in a laminar air flow directed perpendicularly to the ionic winds in the apparatus shown in Figs. 1C and 2C. Such an arrangement is expected not to affect the position of contact between the winds. Considering that the average velocity of the electrospray products at a distance of 20–30 mm from the tip is estimated to be 10–30 m/s (Olumee, Callahan, & Vertes, 1998); Venter, Sojka, & Cooks, 2006, we estimate that ionic winds from two tips separated by a distance of 60 mm will meet in less than 6 ms, which is  40 times smaller than the time needed for the products to reach the aspirator in the vertical laminar air flow. To avoid contamination of the laboratory with the BSA aerosol, we electrosprayed 1% sucrose solution against absolute ethanol as a neutralizing solvent. First, the total concentration of aerosol was measured at different positions by attaching the tube (6) directly to the CPC. As illustrated in Fig. 11, most aerosol particles landed closer to the capillary tip filled with the sucrose solution. This is readily explained by the slower speed of larger sucrose aerosol particles as compared to light ethanol anions. Ignoring losses due to deposition in the connecting tubing, we estimated that approximately 5  107 sucrose nano-particles were generated each second in this experiment. Because no changes in the aerosol concentration were noted after the application of an electrical potential (3–5 kV) to the collecting metal tube (6), we concluded that most aerosol particles were already neutral within 0.25 s. Spectral analysis of aerosol probes taken at different positions revealed a notable difference in the average size and size distribution of the aerosol. The series of spectra presented in Fig. 12 clearly shows that the mass distribution (in mg/m3 units for each diameter interval) shifts to larger diameters in the probes taken farther from the positive capillary filled with the sucrose solution. Both inertial forces and longer neutralization time may be responsible for the longer path of the larger aerosol particles. Thus, the device presented in Fig. 1C may be also used as a separator of generated nano-aerosol particles. 3.6. Retention of enzyme activity. Assuming that the collection efficiency of AP is similar to that of BSA-FITC, we used the fluorescence measurements to calculate the AP content in the samples washed from the chamber walls. AP activity measured in the collected samples was related to the AP content to calculate the specific AP activity, which was then compared to the specific AP activity in the initial AP/FITC-BSA mixture. Table 1 summarizes our results for three independent experiments. One can see that no changes in AP activity were revealed within the accuracy of the measurements. A histogram of the height distribution of AP/BSA-FITC particles measured by AFM showed that 97% of particles have an average height of 1679 nm. Occasionally, particles with heights of 220780 nm (approximately 30 in a scanned area of

2.E+05 C

Counts, cm-3

1.E+05 1.E+05 1.E+05 8.E+04 6.E+04

A

B D

4.E+04 2.E+04 0.E+00

-6 -5 -4 -3 -2 -1 0 1 2 Position, cm

3

4

5

6

Fig. 11. Distribution of sucrose aerosol along the axis between the two capillary tips. Experimental conditions: air flow rate 0.47 0.02 m/s; current 21–24 nA, humidity 30%; aerosol concentration in laboratory air  4  103 cm 3. Left and right vertical lines denote positions of the tips of the capillaries filled with 1% sucrose solution and EtOH, respectively. Points indicated by letters A, B, C and D denote positions for the spectra in Fig. 12.

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Fig. 12. Representative spectra of sucrose aerosol collected under the sucrose tip (panel A); at 2 cm from the tip (panel B); at 3.5 cm from the tip (panel C); and at 4.0 cm from the tip (panel D). See Figs. 1C and 11 for an explanation of the aspirator position with respect to the tip. Table 1 Efficiency of aerosol collection from walls of the aerosol chamber and retention of specific AP activity in aerosol produced by the ESN technique. Parameter/Experiment a

Collected BSA-FITC (%) Specific AP activityb

Expt. #1

Expt. #2

Expt. #3

Average

85 123

86.5 99

86.5 82

867 1 1017 20

a Calculated as the FITC-BSA content in the washing solution compared to the FITC-BSA content in the initial volume of 2.5 mL. ESN was performed at a relative humidity of 22–33%. Total protein concentration in solution was 4.6 mg/mL. b Calculated as the specific AP activity in the dissolved aerosol compared to that in the initial solution.

96  96 mm2 as compared to 1000 particles with the average height of 16 nm) were also observed. These particles may originate from the aggregation of smaller nano-particles or from the occasional drying of large droplets of protein solution. In experiments with urease aerosol, we found 6876% of the electrosprayed urease activity in the dissolved PVP nanofilter when neutralization was performed with ethanol counter-ions. With corona discharge as a source of counter-ions, only 4875% of the urease activity was found in the dissolved filter. Thus, electrospray neutralization provides milder conditions for the generation of biologically active nano-aerosols than neutralization by corona. It is noteworthy that filters were tinged with yellow after the collection of urease nano-aerosol generated with corona neutralization, while they remained white with electrospray neutralization. No color change was noted in control experiments when pure water was electrosprayed against corona discharge. We presume that the nitration of aromatic amino acids (xanthoproteic reaction) is responsible for the color changes observed in the collected urease nano-aerosol. This explanation is supported by the MS data available in the literature, which indicate that a negative corona in the ambient air generates long-lived hydrated NO3 and (NO3 HNO3) anions as major products (Nagato, Matsui, Miyata, & Yamauchi, 2006). Thus, unlike ESN, neutralization with corona-generated counter-ions is accompanied by ‘‘visible’’ chemical modifications of aerosolized protein molecules. It is worth noting that nitrated proteins are considered a major source of allergens in the polluted urban + air (Poschl, 2005). In direct electrospray deposition, highly charged nano-clusters and molecular ions repel each other and quickly settle on a substrate or on the chamber walls. The landing of such multi-charged ions and clusters is expected to liberate quite a lot of energy due to their interaction with induced (mirror) charges on the surface. This energy increases as the square of the charge: it is high enough to break several covalent bonds (Morozov, 2010). In contrast, neutralization of the multicharged macro-ions with mono-charged counter-ions in gas phase and the subsequent landing of neutral nano-clusters and macromolecules onto a solid surface is expected to be much less damaging than in the case of direct electrospray deposition (Morozov & Morozova, 1999a, 1999b). It has already been demonstrated that AP and many other proteins retain their functional activity after electrospray deposition (Avseenko, Morozova, Ataullakhanov, & Morozov, 2002; Bukatina, Morozov, Gusev, & Sieck, 2002; Morozov & Morozova, 1999a, 1999b). Taking into account the published data and the data presented here, we conclude that electrospray neutralization is quite a mild method of atomization which keeps the majority of proteins and other fragile biological molecules intact.

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4. Conclusions We evaluated different factors affecting the concentration and the size distribution of nano-aerosols manufactured by the ESN technique. We demonstrated that solution concentration, humidity, and pressure can be used to control the average size of aerosol particles. We also demonstrated that smaller ESN-generated particles are neutralized more rapidly and closer to the capillary tip from which they were ejected. Such spatial distribution could be used to fractionate the aerosol by size. Because the ESN technique does not employ any specific property of the non-volatile substance (like the ability to evaporate and condense in a gas phase), a great variety of synthetic and natural organic substances and polymers may be turned into nano-particles using this technique. In comparison to other known aerosol generators, the ESN generator described here has a few advantages. First, unlike the De Vilbiss nebulizer and other similar instruments, it does not require compressed air, high-power ultrasound, or a high-speed motor to produce an aerosol. Therefore, it is highly economical, as its power consumption is very low: at a voltage of 10 kV and a current of 1 mA, it consumes only 0.01 W. Therefore, such a generator could operate for many hours on a single AA battery. Second, ESN provides a substantially higher degree of atomization, which reaches an ultimate level when gas-phase solutions of non-volatile macromolecules such as proteins or DNA are produced. It is not known at present whether the new aerosol technique will be applicable for producing aerosols from live cells, spores, or viruses. Both the formation of primary microdroplets and the neutralization process are high-energy processes which might be destructive to living organisms. Based on our previous experiments with the electrospray deposition of proteins and the data presented here, we expect that biological molecules will survive electrospray atomization and subsequent neutralization, and this survival opens a route to simple and economic nano-aerosolizers for effective drug delivery in the treatment of asthma, for neutralization of pathogens in the air, for gas-phase immunization, gas-phase transfer of genes, and many other exciting applications.

Acknowledgement The author gratefully acknowledges support from a DOE grant, DE-F C52-04NA25455. The invaluable help of Dr. T.Y. Morozova in preparing the manuscript and help of Mrs. Jennifer Guernsey in editing text is also enthusiastically acknowledged.

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