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

Contents lists available at ScienceDirect

Journal of Aerosol Science

Journal of Aerosol Science 42 (2011) 341–354

0021-85

doi:10.1

n Corr

Russia.

E-m

journal homepage: www.elsevier.com/locate/jaerosci

Generation of biologically active nano-aerosol by anelectrospray-neutralization method

Victor N. Morozov a,b,n

a National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USAb 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

Article history:

Received 21 October 2010

Received in revised form

16 February 2011

Accepted 16 February 2011Available online 22 February 2011

Keywords:

Electrospray

Nano-aerosol

Neutralization

Enzyme

02/$ - see front matter & 2011 Elsevier Ltd. A

016/j.jaerosci.2011.02.008

espondence address. Institute of Theoretical

Tel.: +7 496 773 0623.

ail address: [email protected]

a b s t r a c t

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.

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 Russianresearchers (Alexandrov et al., 1984; Kozenkov & Fuks, 1976; Zolotoi, Karpov, & Skurat, 1988) demonstrated the feasibilityof 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 micro-droplets, 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 aerosolgeneration techniques, ES atomization stands out as the simplest and most energy-efficient technique capable ofproducing the smallest particles. However, highly charged ES-generated aerosol is intrinsically unstable: the cloud rapidlyexpands due to space charge repulsion, and the charged aerosol particles quickly settle on the walls. To increase aerosolstability, 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 exposedto highly reactive ionization products: radicals, hot molecules, ozone and oxygen atoms; all are destructive for polymerand biological macromolecules as well as for living cells, spores, or viruses. Using atomic force microscopy, the author hasobserved fragmentation of electrospray-deposited polymer molecules upon their neutralization on a mica surface with

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and Experimental Biophysics, Russian Academy of Science, Pushchino, Moscow region 142290,

www.elsevier.com/locate/jaerosci

dx.doi.org/10.1016/j.jaerosci.2011.02.008

mailto:[email protected]

dx.doi.org/10.1016/j.jaerosci.2011.02.008

V.N. Morozov / Journal of Aerosol Science 42 (2011) 341–354342

corona-generated counter-ions (Morozov, 2010). Oxidation and ion-attachment reactions have also been documentedupon 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 toradioactive and corona neutralizers.

The author is aware of only two publications which describe mixing of oppositely charged electrospray-generatedclouds. Camelot, Marijnissen, and Scarlett (1999) reported a rapid mixing of two reagents by coalescence of oppositelycharged microdroplets generated by ES. Solid products precipitated in the coalesced droplets and turned into mono-disperse particles after solvent evaporation. Almekinders and Jones (1999) described the formation of a zero-chargedaerosol 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 hasbeen provided to support such a conclusion.

In our recent papers, we demonstrated that gas-phase neutralization of electrospun polymer nanofibers withES-generated ethanol counter-ions enabled the manufacture of free nanomats (Morozov & Vsevolodov, 2007). Suchnanomats 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-generatedoppositely charged products such as micro- and nano-droplets, small solvent ions, macromolecular ions, nano-clustersof non-volatile substances, and fibers. The presence of the charges enables the outcome of ‘‘mixing’’ the ES-generatedproducts to be controlled by inhibiting the collisions between similarly charged species and accelerating those betweenthe oppositely charged electrospray products.

Here the process of electrospray neutralization (ESN) is studied in more detail to reveal major factors controlling thephenomena of mutual neutralization of ES-generated nano-particles and to characterize how protein molecules survivethis 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-labeledBSA (FITC-BSA), gelatin from bovine skin (type A), polyvinylpyrrolidone (PVP, Mw=360,000), polyvinyl alcohol (PVA withMw=31–50 kDa), polyethyleneimine (PEI, Mw=750,000), glutaraldehyde (GA, 25% solution), TRIS/HCl, NaCl, NaN3, Tween-20, 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, schematicallyillustrated in Fig. 1A, consists of a nearly spherical plastic chamber (1 liter by volume) which has two holes for theintroduction of electrospray capillaries and two hoses for pumping air in and out. One capillary is filled with a solution orsuspension to be transformed into aerosol. The second capillary is filled with a volatile solvent. The solution in eachcapillary is connected to a high-voltage power supply with a platinum wire electrode; the design is described in our recentpapers (Morozov & Morozova, 1999a, 1999b). In addition to the elements just mentioned, the prototype contains threeother features: (i) a fan, (ii) a temperature/humidity sensor (not shown in the schematic of Fig. 1A), and (iii) a port forintroduction of a microscopic slide with a piece of mica attached. The latter was used to collect aerosol particles and fibersfor further analysis with optical and atomic force microscopy. The fan ensured even distribution of the aerosol throughoutthe chamber. The relative humidity was controlled with a commercial digital hygrometer and adjusted by introducing airdried 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 nano-aerosol particles generated in ESN because of the relatively large volume of the chamber which led to substantialaggregation of nano-particles before their deposition on mica. To solve this problem, a smaller ESN chamber was designedas illustrated in Figs. 1B and 2B. With a volume of only 40 mL, it allowed for quick delivery of generated nano-aerosol intoa Scanning Mobility Particle Sizer (SMPS, from TSI Inc., Shoreview, MN). The distance between the capillary tips wasadjustable from 10 to 50 mm. Two glass windows (see image of the cell in Fig. 2B) glued into the cell walls were used toilluminate the ES torch with a laser beam and to observe the ES torch. The cell was connected to a bag filled with filteredair as shown schematically in Fig. 3. Another piece of conductive elastic tubing, 5 mm ID and 10–20 cm long, connected thecell 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 estimateof 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 EScapillary 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

F

+_

Aerosol out

Negativecloud

Positivecloud

To SMPS 1

32

45

5

6

7

To SMPS Air in

+

Air in

Port

Fan

Air in

_

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.

V.N. Morozov / Journal of Aerosol Science 42 (2011) 341–354 343

Compressed air

1

1

2

3

4 5 5

6 7

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 awide (70 mm in diameter) hole in the bottom plate. A porous tissue (4) covered the top of the cell, protecting it fromoccasional air motions. The chamber had two portholes (5), 120 mm from the bottom, used for introduction of capillaryholders. Another porthole (7), placed 20 mm above the bottom, was used to introduce a metal tube (6) (4 mm ID) foraspiration of aerosol at different positions with respect to the capillary tips. The vertical air flow created by the fan wasmeasured with an air flow meter (Testo 415, GmbH & Co., Germany). The fan created a downward air velocity of0.4070.02 m/s, which at the level of the capillaries was uniform within a radius of 3.5 cm around the axial line of thecylinder jar. As aspiration of air into the tube (6) at a volume rate of 10 mL/s was much lower than the overall volumetricdownward flow rate exceeding 1.5 L/s, we expected that aspiration would not disturb the flow distribution in the chambersignificantly. Aerosol particles and neutralizing ions moved downward to the aspiration tube in �0.25 s. A conductiverubber 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 (consuming10 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 4300 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 small-volume 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 conductiveelastic 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 certainhumidity. To avoid problems with dew formation in the SMPS, the air flow at the output of the ESN chamber was mixedwith an equal flow of purified air dried over silica gel. Thus, when the air in the ESN chamber was 100%, the humidity ofthe 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 viaa 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 ofthe 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 ofhumidity effects, the sensor of a digital hygrometer was introduced into the bag (3) schematized in Fig. 3, and the air inside thebag was dried by passing the filtered air through a silica gel column or humidified by introducing strips of Whatman paperwetted 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 AFMmeasurements. Tygon tubing was attached close to the scanning head in order to direct a weak jet of air dried over silicagel onto the scanning cantilever and the substrate surface. Keeping the environment dry prevents formation of a waterbridge between the tip and the mica surface and eliminates the effects of humidity. A tapping mode with a resonancefrequency of 300–350 kHz was used in all scanning experiments. Tip quality was routinely controlled by scanning samplesof electrospun PVP solutions which presented a variety of fibers from sub-nanometer height to 500 nm (Morozov andMorozova, 1998). Cantilevers which did not reveal fibers 1 nm high were discarded.


In a typical experiment, mica was pre-treated in a 1% solution of poly(ethyleneimine) to enhance binding of proteinnanostructures to the surface. Protein aerosol generated in the chamber (Figs. 1A and 2A) was allowed to settle on the micasurface for a few minutes. Then the sample was exposed for 2–5 min to glutaraldehyde (GA) vapor to prevent moleculesand clusters from displacement by the cantilever tip upon scanning. To decrease water activity in the 25% commercial GAsolution, dry NaCl powder was added to the solution in large excess. The relative humidity over this NaCl slurry wasreduced to �77%. The procedure of GA fixation at such moderate humidity prevented protein molecules and clusters fromchanging shape as a result of interacting with the solid surface. Gelatin nano-aerosol particles and fibers were collected for5 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 anelectric conductivity of s=10–13 mS cm�1. The protein concentration was determined using an extinction coefficient of280 nm according to Landt, Boltz, and Butler (1978). The AP solution, with a protein concentration of 1.470.1 mg/mL, wasmixed with an equal volume of dialyzed FITC-labeled BSA solution with a protein concentration of 3.2 mg/mL. The latterwas measured using a quartz crystal microbalance (Morozov & Morozova, 1999a, 1999b). Microliter aliquots of thismixture 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 DualChannel Fluorometer (Turner BioSystems, Sunnyvale, CA). A graph of the fluorescence vs. added volume of the AP/BSA-FITCmixture 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 theaerosol generator chamber shown in Figs. 1A and 2A. A second capillary was filled with �10 mL of absolute ethanol. Apositive pole of a high-voltage power supply (6–8 kV) was connected to the platinum wire inside the first capillary, whilethe negative pole was connected to a similar electrode in the second capillary, resulting in a steady current of 50720 nA.The distance between the capillary tips (30–50 mm in OD) was set to 60–80 mm. To accelerate the process, a pressure of18–20 cm of water was applied to the capillary with the AP/BSA-FITC mixture. The initial air humidity in the chamber was22%. The entire volume of the mixture was aerosolized in 5–6 min, increasing the humidity to 33%. The chamber was keptclosed 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 theblocking 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 thebuffer was collected for further analysis. The fluorescence of the collected solution was measured and used to determinethe efficiency of aerosol collection.

To compare specific AP activity before and after electrospraying, 10 mL probes of the initial AP/BSA-FITC mixture diluted1:1000 with the blocking solution and 10 mL probes of the solution collected after the chamber washing were each addedto 1 mL of commercial pNPP substrate, and the rates of substrate hydrolysis were measured by monitoring changes in theoptical density at 405 nm. Each rate was then divided by the fluorescence intensity of the respective probe to account forthe enzyme loss in the collected sample. The ratio of the normalized enzymatic activity in the collected sample and thenormalized initial activity of this enzyme was used as a measure of retention of specific enzyme activity in theaerosolized 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 ureasesolution (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 distancebetween the capillary tips was set to 100 mm. The corona ionizer was placed in the same position as the capillary tip filledwith 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 airpumped from the chamber was replaced with fresh air filtered through a Whatman HEPA-VENT capsule (Cole-ParmerInstrument Company, Vernon Hills, IL) connected to the second hose in the chamber. After electrospraying 2–4 mL of theurease solution over 1870.5 min at a steady current of 200720 nA, the PVP filter was dissolved in 20 mL of water. Thissolution was added to 0.5 mL of a substrate solution: 0.15 M urea dissolved in 5 mM phosphate buffer, pH=7.6. Changes inpH due to decomposition of urea by added urease were registered by a chart recorder and used to calculate the enzymereaction rate. The calculated rate was compared to the urease activity in a similar volume of the initial urease solutionused 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 adistance equal to the distance between the capillary tips. This is explained by the two large potential drops (in the vicinity

I. Standard electrospray

II. Electrospray with neutralization Dry neutral residue of

mother drop

Dry residue of mother and daughter drops

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 asTaylor cones become visible on both capillary tips, and a stable current goes through the capillaries, provided the chamberis not touched by a hand or any conductive object. One may thus conclude that the electrospraying process in the ESN isentirely controlled by a local electric field at each tip and that the electrospraying proceeds exactly as in the ESD and ESI-MS 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 andnegatively charged products are generated at equal rates, thereby producing a bipolar ‘‘plasma’’ which undergoesneutralization in parallel with the decomposition of charged microdroplets. It is well known that a jet of electrosprayedmother droplets emitted from the capillary tip is transformed into a cloud of highly charged dry residues of progeny nano-droplets 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 areneutralized by giving or accepting electrons from a conductive substrate. Thus, in electrospray deposition, theneutralization process is completely separated from the atomization process. In ESN, these two processes occursimultaneously in the gas phase and can affect each other. One may expect, for example, that larger aerosol particleswill be produced in the ESN, if neutralizing counter-ions reach the charged aerosol microdroplets before the latterexperience the full set of drying-decaying cycles. In the ultimate case, when neutralization of mother droplets happensbefore the first electrostatic decay, as illustrated in the lower part of Fig. 4, all the non-volatile content of the motherdroplet will end up in a single dry residue particle. One may expect from such scenarios that any conditions which reducethe speed of solvent evaporation from the mother droplet will favor the formation of larger particles. Because of thiscompetition between neutralization and electrostatic decay of droplets, the size distribution of the generated particlesshould strongly depend on the rate of solvent evaporation from the mother droplet and on the mobility of the counter-ions. Since at higher humidity ES-generated microdroplets evaporate more slowly, they have a good chance of becomingneutralized before reaching the Raleigh limit. At low humidity, the droplet has a chance to disintegrate beforeneutralization 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 spectraobtained 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 particlesgenerated at moderate humidity have heights lower than 30 nm, many particles with larger heights were observed onthe mica surface when ESN was performed at high humidity. This difference cannot be explained by a difference in theflattening of the PVA particles in contact with the mica surface at different humidities, since at high humidity thehydrophilic PVA particles should be more prone to deformation and should acquire a flatter shape in contact withthe surface than at moderate humidity. We conclude that larger aerosol particles are generated at higher humidity in goodaccordance with the scenario schematized in Fig. 4.

The ESN process with the same PVA dissolved in NN-dimethylformamide (DMFA) resulted in notably larger particles ascompared with those generated at the same low humidity from an aqueous solution of PVA of similar concentration. Onecan see from the histograms of PVA particles presented in Fig. 6 that fewer small particles and more large particles weregenerated from the solution in DMFA than in water under otherwise identical conditions. This difference can be readily

05

1015202530354045

5Particle height, nm

Part

icle

s, %

98%

75%

15 25 35 45 55 65 75 85 95 105

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, 7575%). The aerosol was generated in the chamber depicted in Fig. 1A. Absolute ethanol was used to generate negative

counter-ions.

05

1015202530354045

5 25 45 65 85 105

125

145

165

Heights, nm

% o

f par

ticle

s

In water

In DMFA

Fig. 6. Comparison of the height distribution of nano-aerosol particles generated from 1% PVA solution in water (white bars) and in DMFA (black bars) at

22% humidity.


explained by the notably higher boiling point of DMFA (153 1C) and, hence, the slower evaporation rate of DMFA from themother droplets as compared to that of water. The slower evaporation rate increased the probability of neutralization ofthe mother droplets before electrostatic decay in good accordance with the ESN scenario shown in Fig. 4. Of course, part ofthe difference may originate from the difference in the electrospraying process for these two solvents, which have differentdensities, 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 leastthree ways: (i) due to changes in the particle shape in contact with the substrate surface, as discussed above, (ii) due toaerosol aggregation before landing, and (iii) due to differences in the diffusion-controlled deposition rates for particles ofdifferent sizes. While the second factor is expected to decrease the fraction of observed small particles, the third one leadsto underrepresentation of larger particles in the AFM images. These limitations of the AFM technique do not affect thequalitative conclusions drawn above regarding the effects of humidity in ESN. The conclusions were further supported byspectra obtained with the SMPS device.

The humidity dependence of aerosol spectra was also evaluated by the SMPS device using 1% solutions of both sucroseand dialyzed BSA. The results for the sucrose aerosol are presented in a series of panels in Fig. 7. In accordance with ourexpectations, the spectrum of the nano-aerosol obtained by ESN at a very low humidity (panel A in Fig. 7) is drasticallydifferent from those obtained at moderate and high humidities. The presence of a large fraction of small nano-particleswith a broad distribution of sizes indicates that at low humidity, the mother droplets had a chance to decompose andproduce 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-dispersenano-aerosol with a major peak at 20 nm was produced. The latter size still cannot be attributed to the dry residue of theentire mother droplet, since the dry residue with a diameter of 20 nm would require evaporation of a 1% sucrosemicrodroplet with a diameter of 130 nm—an order of magnitude smaller than the average size estimated for the motherdroplets (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

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 sucrosedroplets 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 withneutralization by corona products from the sharp platinum needle described above. The humidity dependence of aerosolsizes was similar to that observed in neutralization with EtOH counter-ions. One may thus conclude that the motherdroplets undergo some Coulomb fission even at 100% humidity. It may happen near the Taylor cone as a result ofdestabilization 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 ofproducing occasional droplets (spills). To avoid these, we controlled the flow rate of solutions through the capillary (in therange of 0.2–0.8 mL/min), not by a pump but by applying a hydrostatic pressure, which increased the flow rateproportionally to the applied pressure and which provided a stable cone-jet electrospray at combinations of flow rateand 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 ispresented in Fig. 8. It contains two peaks: at 30 nm and at 150 nm. Though the concentration of the former is �10 timesgreater than that of the large particles (compare Figs. 8C and B), they accounted for only �10% of the BSA content in thetotal 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 ofsmaller nano-clusters (peak at 12–30 nm) of BSA and a ten-fold increase in the rate of their generation. The larger fractionof nano-particles (with a mean diameter ranging from �100 nm at low humidity to �150 nm at high humidity) exhibitedfewer changes in the diameter, but the rate of their generation increased �10-fold when a pressure of 5000 Pa wasapplied. Humidity does not notably affect the pressure dependence of the nano-aerosol diameter and the rate of itsgeneration as one can see from comparison of Fig. 8B and D. Of course, the numbers presented in Fig. 8 will vary with thediameter of tip and the length of the extended thin part of the capillary. The effect of applied pressure just described issimilar to the way pressure affects the formation of charged droplets: pressure applied to the solution in the capillarymakes it spray more rapidly, resulting in a larger number of microdroplets with larger sizes being generated in the ESNprocess (Fernandez de la Mora, 2007). A practically important result of studying the effects of pressure consists of theability to greatly vary the rate of nano-aerosol production without substantially changing the nano-aerosol spectrum—aphenomenon which could be exploited in nano-aerosol generators for medical purposes to control inhaled doses.

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 80720 nm at pressure p=0 cm water to 115715 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 6673%. 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 85Kr neutralizer (A) and without the neutralizer (B). ESN at

the relative air humidity of 32%.


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 of2.370.6 nm (ESN at 45% humidity in the chamber in Fig. 1A) and rare particles 20–30 nm high. The former heightcorresponds to approximately one half of the diameter expected for a single PVA molecule with a molecular mass of31–50 kDa collapsed into a spherical ball. It is worth noting that a similar reduction in the height of collapsed polymerglobules has been reported for PVA and polyethylene glycol molecules after ES deposition onto a mica surface (Morozovand Morozova, 1998). We may conclude from such similarity in the deformation of neutral and highly charged globulesthat it is not electrostatic forces resulting from mirror charges but rather direct interaction with the surface that isresponsible for the observed flattening of collapsed hydrophilic polymers which lack a stable internal structure. Aerosolparticles 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 adiameter of 6.3–6.7 nm which may be attributed to nano-aerosol particles comprised of single BSA molecules with theX-ray sizes of the BSA molecule (5.5�5.6�12.0 nm3) according to Carter and Ho (1994). This spectrum corresponds wellto the data obtained in the AFM analysis, which showed a notable fraction of particles with average heights of 6.472.7 nmwhen BSA aerosol was generated at 34% humidity from 1% solution. In addition to these particles, larger particles with AFM

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 57712 nm were also observed. Particles with heights of 5.773.1 nm were observed after electrospraying ofdiluted solutions of ovalbumin (ESN of 0.1% solution at 75% humidity in the chamber), close to the average size of theovalbumin molecule 7�4.5�5 nm3 according to X-ray analysis (Stein, Leslie, Finch, & Carrell 1991). Thus, ESN of dilutedprotein solutions produces a nano-aerosol comprised of single protein molecules as the major ESN product. The closeagreement between the AFM height and the X-ray dimensions of the molecules thus deposited indicates that themolecules retain their size, shape, and structure upon landing. We envisage that this mild technique may be used toprepare 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 afibrillar protein, gelatin, at a relatively high concentration and low humidity (A=20–30%) resulted in formation of aerosolcomprised of nanofibers and nano-loops rather than spherical nano-clusters. Similar fibrillar structures have beenobserved on the mica surface after direct ES deposition (Morozov & Morozova, 1998). However, in the ESN process it takesmuch longer (minutes, rather than the milliseconds of ESD) for neutral products to reach the mica substrate by diffusion. Itseems remarkable that dry neutral gelatin nanofibers with a diameter of only 2–10 nm as determined by the AFM heightwere rigid enough to keep their shape in dry air for such a long time, even though such a form is expected to be highlyunfavorable 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 spectrapresented in Fig. 9A, in addition to the monomolecular aerosol particles, a broadly distributed peak of BSA nano-clusterswith 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 nmclusters were still readily observable. One may thus conclude that the small protein ions completely lost all their chargeswithin the few seconds it took to transfer the ESN-generated nano-aerosol into the SMPS device. Another explanation forthe disappearance of single-molecule nano-aerosol particles involves assuming that these reach the SMPS bearing multiplecharges, which makes them too mobile to be detected in the electrostatic classifier. In contrast to the single-moleculeparticles, large BSA nano-clusters were readily observable without neutralizer, and their concentration was nearlyidentical to that with the neutralizer, as is seen when comparing the two spectra presented in Fig. 9A and B. Theoreticalanalysis indicates that �9.2% of the particles with a diameter of 22 nm passing through the radioisotope bipolarneutralizer acquire one positive charge and only 0.02% acquire two positive charges (see the operating manual of Model3936 SMPS). Taking into account that the SMPS software accounts for this charging ratio, and comparing the 22 nm peakintensities in the two spectra in Fig. 9, we concluded that �10% of the 22-nm BSA nano-particles retained one residualpositive charge for at least 0.4–4 s after being generated in the ESN process. The quantitative similarity in the 22 nm peaksobtained with and without neutralizer allow us to think that the final stage of neutralization in ESN provides conditionsidentical to those in the bipolar neutralization (Scalf et al., 1999; Frey et al., 2005). Since initially not only nano-clusters,


but also hydrated protons, Na+, and other ions comprise the positive cloud, it is possible that the late stages in ESN processmay resemble bipolar neutralization, with both positive and negative high-mobility ions playing the same role as inbipolar charging. Small positive and negative ions move faster than larger charged nano-clusters. Therefore, the latter mayarrive to the scene where small ions already have formed a bipolar mixture and where incoming charged nano-clustersmay acquire an equilibrium charging state. These hypotheses about the mechanisms of neutralization have to be verifiedexperimentally, 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 inducedby the motion of electrospray products and counter-ions. Where between the two capillary tips do those two ionic windsmeet? To answer this question, we studied neutralization under conditions which prevented the mixing of newly formedproducts with previously formed products. This was achieved by performing neutralization in a laminar air flow directedperpendicularly to the ionic winds in the apparatus shown in Figs. 1C and 2C. Such an arrangement is expected not toaffect the position of contact between the winds. Considering that the average velocity of the electrospray products at adistance 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 absoluteethanol as a neutralizing solvent. First, the total concentration of aerosol was measured at different positions by attachingthe tube (6) directly to the CPC. As illustrated in Fig. 11, most aerosol particles landed closer to the capillary tip filled withthe sucrose solution. This is readily explained by the slower speed of larger sucrose aerosol particles as compared to lightethanol 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 concentrationwere noted after the application of an electrical potential (3–5 kV) to the collecting metal tube (6), we concluded that mostaerosol 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 sizedistribution 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 withthe sucrose solution. Both inertial forces and longer neutralization time may be responsible for the longer path of thelarger aerosol particles. Thus, the device presented in Fig. 1C may be also used as a separator of generated nano-aerosolparticles.

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 tocalculate the AP content in the samples washed from the chamber walls. AP activity measured in the collected sampleswas related to the AP content to calculate the specific AP activity, which was then compared to the specific AP activity inthe initial AP/FITC-BSA mixture. Table 1 summarizes our results for three independent experiments. One can see that nochanges 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 anaverage height of 1679 nm. Occasionally, particles with heights of 220780 nm (approximately 30 in a scanned area of

0.E+002.E+04

4.E+04

6.E+04

8.E+04

1.E+05

1.E+05

1.E+05

2.E+05

-6Position, cm

Cou

nts,

cm

-3

B A

C

D

-5 -4 -3 -2 -1 0 1 2 3 4 5 6

Fig. 11. Distribution of sucrose aerosol along the axis between the two capillary tips. Experimental conditions: air flow rate 0.470.02 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.

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 1Efficiency of aerosol collection from walls of the aerosol chamber and retention of specific AP activity in aerosol produced by the ESN technique.

Parameter/Experiment Expt. #1 Expt. #2 Expt. #3 Average

Collected BSA-FITCa (%) 85 86.5 86.5 8671

Specific AP activityb 123 99 82 101720

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 mayoriginate 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 nano-filter 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 milderconditions for the generation of biologically active nano-aerosols than neutralization by corona. It is noteworthy thatfilters were tinged with yellow after the collection of urease nano-aerosol generated with corona neutralization, while theyremained white with electrospray neutralization. No color change was noted in control experiments when pure water waselectrosprayed 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 MSdata available in the literature, which indicate that a negative corona in the ambient air generates long-lived hydratedNO3�

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 aerosolizedprotein molecules. It is worth noting that nitrated proteins are considered a major source of allergens in the polluted urbanair (P +oschl, 2005).

In direct electrospray deposition, highly charged nano-clusters and molecular ions repel each other and quickly settleon a substrate or on the chamber walls. The landing of such multi-charged ions and clusters is expected to liberate quite alot of energy due to their interaction with induced (mirror) charges on the surface. This energy increases as the square ofthe charge: it is high enough to break several covalent bonds (Morozov, 2010). In contrast, neutralization of the multi-charged macro-ions with mono-charged counter-ions in gas phase and the subsequent landing of neutral nano-clustersand macromolecules onto a solid surface is expected to be much less damaging than in the case of direct electrospraydeposition (Morozov & Morozova, 1999a, 1999b). It has already been demonstrated that AP and many other proteins retaintheir 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 datapresented here, we conclude that electrospray neutralization is quite a mild method of atomization which keeps themajority of proteins and other fragile biological molecules intact.


4. Conclusions

We evaluated different factors affecting the concentration and the size distribution of nano-aerosols manufactured bythe ESN technique. We demonstrated that solution concentration, humidity, and pressure can be used to control theaverage size of aerosol particles. We also demonstrated that smaller ESN-generated particles are neutralized more rapidlyand closer to the capillary tip from which they were ejected. Such spatial distribution could be used to fractionate theaerosol by size. Because the ESN technique does not employ any specific property of the non-volatile substance (like theability to evaporate and condense in a gas phase), a great variety of synthetic and natural organic substances and polymersmay 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, unlikethe De Vilbiss nebulizer and other similar instruments, it does not require compressed air, high-power ultrasound, or ahigh-speed motor to produce an aerosol. Therefore, it is highly economical, as its power consumption is very low: at avoltage of �10 kV and a current of �1 mA, it consumes only 0.01 W. Therefore, such a generator could operate for manyhours on a single AA battery. Second, ESN provides a substantially higher degree of atomization, which reaches an ultimatelevel 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 processeswhich might be destructive to living organisms. Based on our previous experiments with the electrospray deposition ofproteins and the data presented here, we expect that biological molecules will survive electrospray atomization andsubsequent neutralization, and this survival opens a route to simple and economic nano-aerosolizers for effective drugdelivery in the treatment of asthma, for neutralization of pathogens in the air, for gas-phase immunization, gas-phasetransfer 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 enthusiasticallyacknowledged.

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