LANG l\.A u I R----_______jll'~tfi@M
`
`I V I
`
`pubs.acs.mg/Lanwnuir
`
`Cleaning with Bulk Nanobubbles
`. Paul M. J. Terpstra/ Guangming Liu,*'1
`
`Jie Zhu, 1. Hongjie An/ Muidh Alheshibri/ Lvdan Liu, 1
`.
`and Vincent S. ]. Craig*"0
`
`tDepartment of Chemical Physics, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and
`Technology of China, Hefei, PR China 230026
`*Department of Applied Mathematics, Research School of Physical Sciences and Engineering, Australian National University,
`Canberra ACT 2601, Australia
`§Consumer Technology Research Institute, vVageningen, The Netherlands
`
`~ Supporting Information
`
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`ABSTRACT: The electrolysis of aqueous solutions produces solutions that are
`supersaturated in oxygen and hydrogen gas. This results in the formation of gas bubbles,
`including nano bubbles ~ 100 nm in size that are stable for ~24 h. These aqueous solutions
`containing bubbles have been evaluated for cleaning efficacy in the removal of model
`contaminants bovine serum albumin and lysozyme from surfaces and in the prevention of
`the fouling of surfaces by these same proteins. Hydrophilic and hydrophobic surfaces were
`investigated. It is shown that nanobubbles can prevent the fouling of surfaces and that they
`can also clean already fouled surfaces. It is also argued that in practical applications where
`cleaning is carried out rapidly using a high degree of mechanical agitation the role of
`cleaning agents is not primarily in assisting the removal of soil but in suspending the soil
`that is removed by mechanical action and preventing it from redepositing onto surfaces.
`This may also be the primary mode of action of nanobubbles during cleaning.
`
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`INTRODUCTION
`
`N anobubbles or fine bubbles are small gaseous entities that are
`found on surfaces and in
`the bulk when solutions are
`supersaturated with gas. V They were first proposed to explain
`unusual long-range attractive forces observed between hydro(cid:173)
`phobic surfaces3 and later imaged on surfaces using atomic
`force microscopy.4
`5 They are of particular interest as the
`'
`observed stability of nanobubbles is inconsistent with current
`theories of bubble dissolution.6
`7
`l\tioreover, surface nano(cid:173)
`'
`bubbles exhibit exceptionally high contact angles that cannot
`currently be explained.3 Despite the theoretical challenges in
`understanding nanobubbles, they are already findin& a~plica­
`tions in a broad range of areas including medicine,.,_ 1 crop
`3 and bioremedia(cid:173)
`production, u. controlling bounda1y slip, J
`tion.14 The possible advantages of using nanobubbles in a broad
`range of applications include the ease with which they can be
`produced, the low cost of materials, and the potential to easily
`remove them from the system once they have performed their
`function. Additionally, they may provide solutions to industrial
`challenges with low environmental impact. It is for this reason
`that nanobubbles are seen as promising agents for cleaning.
`Cleaning in a scientific sense is often associated with
`detergency, but common experience tells us that cleaning does
`not require the presence of a surfactant or detergent if sufficient
`mechanical means is employed. Our prior work, as well as the
`work of others, describes the prevention of both fouling and
`17 The role of nano(cid:173)
`cleaning using surface nanobubbles. 15
`-
`bubbles in preventing fouling is to provide a mechanical barrier
`to the adsorption of material on the surface. Thus, the regions
`
`V ACS Publications
`
`© xxxx American Chemical Society
`
`A
`
`of the surface that are decorated with nanobubbles mask the
`surface from exposure to contaminants. VVhen employed in this
`manner, the regions of the surface not covered by nanobubbles
`will still be fouled, so this is not a preferred mode of operation.
`However, it was shown that the production of nanobubbles on
`a surface that is already contaminated results in surface cleani_ng
`that can result in nearly all contamination being removed. 1
`'"' L'
`Nanobubbles in these studies were prepared by electrolyzing
`water, with the resultant supersaturation of dissolved gas
`leading to the formation of surface nanobubbles. As
`the
`nanobubbles expanded, the advancing three-phase line, which
`marks the bounda1y between the nanobubble and the liquid on
`the surface, removed the contaminant from the surface. It was
`found that cleaning was more effective with nanobubbles than
`with sodium dodecylsulfate (SDS) surfactant that is commonly
`used in detergents and that cleaning was further enhanced
`
`when surfactant was also used. 16' l'.7 Surfactants clean by
`reducing the adhesive forces between the contaminant and
`the surface, whereas surface nanobubbles clean when the
`advancing three-phase line transfers contaminants from the
`substrate to the gas/solution interface. The high energy of the
`interface provides for powerful cleaning action and reflects the
`comparative strength of capillary forces compared to surface
`
`Special lssm~: Nanobubbles
`
`Received:
`Revi§ed:
`
`March 15, 2016
`April 22, 2016
`
`CONFIDENTIAL - ATTORNEYS' EYES ONLY
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`TC00124352
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`

`

`Langrnulr
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`involves a
`forces. Thus, cleaning with nanobubbles also
`mechanical component, in addition to surface energetics. A
`particular advantage of using nanobubbles for cleaning is that
`there is no need to rinse surfactant residue from the surface
`after cleaning has been effected. Thus, nanobubbles have been
`proposed for use in specialist applications such as cleaning
`semiconductors. rn Furthermore, cleaning with nanobubbles has
`the potential advantages of reducing the considerable environ(cid:173)
`mental impact of surfactant use,
`lowering the use of
`nonrenewable resources, and lessening emissions into the
`environment.
`Electrolysis has also been reported to produce nanobubbles
`9
`25 Electrolysis results in the supersaturation of
`in the bulk. l
`-
`oxygen and hydrogen in the anodic and cathodic streams of the
`solution, respectively, causing bubbles to be nucleated. Larger
`bubbles quickly rise to the surface and burst, but particle sizing
`techniques show persistent nanoparticles of around 100 nm
`following electrolysis that are not present in control solutions.
`A number of authors have concluded that the nanoparticles
`produced by electrolysis are gaseous, as described below. This is
`consistent with the range of methods used for their production,
`which all involve supersaturation and the absence of nano(cid:173)
`particles in the control solutions that have been investigated.
`Kikuchi et al."'3 electrolyzed water to generate solutions
`supersaturated ·with oxygen. The formation of nanoparticles in
`these solutions was detected by light scattering, and their
`concentration was analyzed by measuring the oxygen content.
`The dissolved oxygen level was analyzed with a dissolved
`oxygen (DO) meter and the Winkler titration method. 26 It was
`shown that these methods access only the dissolved oxygen, not
`that present within bubbles, but by the addition of sulfuric acid,
`the stability of the nano bubbles was disrupted and a subsequent
`Winkler titration revealed an increase in oxygen concentration.
`The difference in oxygen concentration was used to determine
`the concentration of bulk nanobubbles of oxygen produced
`during electrolysis. Similarly, in previous work Kikuchi et al. 19
`investigated solutions supersaturated in hydrogen gas. By
`comparing the results from a dissolved hydrogen meter and
`chemical analysis,
`they found that the chemical analysis
`revealed a higher concentration of hydrogen gas and attributed
`the difference to the existence of hydrogen nanobubbles. Note
`that the chemical analysis required acidification. This is thought
`to remove the charge on the nanobubbles and destabilize them.
`The production of bulk nanobubbles by mechanical methods
`of supersaturation has also been reported. Ohgaki et al.~\;·
`reported that solutions of bulk nanobubbles consisting of
`nitrogen, methane, and argon with an average radius of SO nm
`were stable for 2 weeks. They were produced by mechanical gas
`injection, which leads to a supersaturated solution. A scanning
`electron micrograph of replica films of rapidly frozen and
`fractmed solutions showed a SO-nm-radius cavity. Ushikubo et
`aI.2'; reported the production of both oxygen and air-filled
`nanobubbles by mechanical methods. The size of the
`nanobubbles was characterized by dynamic light scattering.
`Additionally,
`it was claimed that nanobubbles caused an
`increase in the Tl relaxation time of the solutions as measured
`by NMR. Kobayashi et al.29 have used an Archimedes
`microelectromechanical sensor for
`the detection of nano(cid:173)
`bubbles. This technique is sensitive to the density of particles;
`therefore, particles that are positively buoyant are readily
`distinguished from negatively buoyant particles. Furthermore,
`the size of the particle is measured independently of the
`density. Using this technique, nanobubble solutions showed
`
`•••
`
`positively buoyant particles of the same size (~ 100-250 nm)
`as particles detected by dynamic light scattering, providing
`strong evidence for the existence of bulk nanobubbles. ·when a
`modulation interference microscope is used, particles can be
`distinguished on the basis of the refractive index and thereby
`bubbles (refractive index = 1) can be distinguished from other
`particles. Bunkin et al. have identified stable nanobubbles in the
`size range of 2S0-750 nm using this technique. 30
`Temperature changes have also been used to produce bulk
`nanobubbles 290 nm in diameter. 1
`i Increasing the temperature
`reduces the solubility of dissolved gases, resulting in super(cid:173)
`saturation. These experiments were performed with the
`temperature change being induced in a cell mounted in a
`light-scattering instrument. The conclusion is that particles
`produced upon increasing the temperature are nanobubbles as
`the solubility of most materials other than gas will increase with
`increasing temperature.
`Here we test the hypothesis that bulk nanobubbles produced
`through the electrolysis of aqueous electrolyte solutions
`enhance the cleaning capacity of the solution. Electrolysis
`results in the production of hydrogen and oxygen gas as well as
`a range of other chemical reactions. To control for the effect of
`chemical changes as opposed to physical changes in the
`solutions, we compare solutions that have been filtered to
`remove nanobubbles with those containing nanobubbles in
`order to determine the effectiveness of the nanobubbles as
`cleaning agents. Additionally, we use untreated solutions as
`control samples.
`
`iiili MATERIALS AND METHODS
`Sodium chloride solutions of concentration 2.1 X 10-3 M were
`prepared with water that had been purified using either a Milli-Q
`gradient or an ELGA purelab Chorus 3 system, resulting in a solution
`with conductivity of ~250 11s/cm. Nanobubbles were produced by
`electrolysis using an ec-H20 Nanoclean cell
`that employs five
`electrode plates of platinum with a plate spacing of 2.79 mm. The
`device and control electronics is a proprietry design of Tennant
`Company. Aqueous solution was treated electrochemically using a cell
`voltage of ~24 Vat a flow rate of 7.5 mL s- 1
`• This results in the fluid
`transiting the plates in approximately 13 s. The electrolysis reaction
`produces m.-ygen and hydrogen gas in a molar ratio of 1:2 according to
`the reaction equations shown in Figure l.. Immediately after treatment,
`numerous visible bubbles are apparent in the solution. Because of
`buoyancy, the larger bubbles rise to the surface and burst, hence the
`appearance of the solution changes significantly during the first few
`minutes after production. Filtering was conducted on selected samples
`using syringe filters of either a 20 nm (0.02 µm Whatman Anotop 10
`inorganic with an Al20 3 membrane) or 450 nm (0.45 µm l\fillipore
`Millex-LCR Hydrophilic PTFE membrane) pore size.
`For cleaning studies, we developed a protocol that could be used to
`determine if any cleaning observed was due to electrolysis and
`furthermore could determine the size of the species responsible for
`such action. This was specifically developed to enable any cleaning due
`to chemical changes that occur during electrolysis to be separated from
`cleaning associated with the nanoparticles produced during
`electrolysis. Note that this is necessary because other reactions will
`occur in the presence of chloride ions, including the formation of
`hypochlorous acid. In addition the efiluent from the cathode and
`anode locally will be alkaline and acidic, respectively. It is known that
`alkalinity in particular can affect cleaning performance. However, the
`streams are mixed, and the change in pH of the solution upon
`electrolysis is minimal by the time the solutions are used. Five
`solutions were studied as depicted in T:1ble 1.
`A NanoSight (NS300, Nanosight, software Nanosight V3.1)
`instrument for nanoparticle tracking analysis using a blue laser light
`source (70 mvV, ;[ = 405 nm) at 25 °C was used to size the
`
`B
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`XXX-XXX
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`TC00124353
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`

`Langrnulr
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`Ekdro!y?>i~'>
`Reduction (at !he cathi::.O:e)
`4H,.Dd)"4a -2H;{g) + 40Hfaq}
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`Oxidation iat ihe anodf.l)
`2H;Otn- O,.(gi+ 4W(aq)+4f.>·
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`Chern.II
`2H;0(!) ~ 2H)g)+Oz(gl
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`E!ii-ctrnlf$b C@·I!
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`
`Figure 1. Schematic showing the production of samples for
`investigation. The electrolysis of 2.1 mM NaCl solutions is used to
`produce a solution supersaturated with oxygen and hydrogen gases
`(ec-H20), which is used as a test solution. Filtration through a 450 nm
`filter excludes all particles and bubbles larger than 450 nm and
`produces a second test solution (ec-H20 sub 450 nm). Filtration
`through a 20 nm filter removes the nanoparticles produced during
`electrolysis and provides a control solution in which the chemical
`effects of electrolysis are maintained (ec-H20 sub 20 nm). The
`untreated electrolyte sample, filtered or unfiltered, was also used as a
`control solution.
`
`Table 1. Nomenclature for the Solutions Employed to
`Investigate Cleaning
`
`designation
`
`NaCl(aq)
`NaCl(aq) sub
`450 nm
`ec-H20
`ec-H10 sub
`450 nm
`ec-H20 sub
`20 nm
`
`description
`
`purified water with 2.1 mlvI NaCl added
`purified water \Vith 2.1 mJvI NaCl added that has been filtered
`through a 450 nm filter
`purified water with 2.1 ml'vi Na Cl that has been electrolyzed
`purified water with 2.1 mM NaCl that has been electrolyzed
`and then filtered through a 450 nm filter
`purified water with 2.1 mM NaCl that has been electrolyzed
`and then filtered through a 20 nm filter
`
`nanoparticles and determine the concentration. Each measurement
`comprised an analysis of five movies, each 60 s long, captured at 25
`frames/s. The camera level was set to 14, the threshold to 3, the gain
`to 366, and the shutter to 31.48 ms, and analysis was conducted using
`a solution viscosity of 0.888 cP. The Nanosight determines the size of
`individual particles from their diffusion under Brownian motion using
`nanoparticle tracking. The concentration is directly determined by
`counting the number of particles observed in a known volume. The
`tracking of numerous particles enables the size distribution of particles
`to be determined. Samples of ec-H20 were filtered through a 450 nm
`filter before introduction into the NanoSight measurement cell, to
`ensure that larger bubbles did not interfere with the measurements. A
`Zetasizer Nano ZS (l'vlalvern) was also used to measure the size of
`nanoparticles by dynamic light scattering as well as the zeta potential
`by laser Doppler microelectrophoresis in an ac electric field.
`Proteins bovine serum albumin (BSA), sourced from Hualvyuan
`Biotechnology Co., and lysozyme, sourced from Bio Basic Int., were
`used as model contaminants. The amount of material was measured
`using spectroscopic ellipsometry (M2000 V, J. A. Woollam, U.S.A.) at
`two incident angles of 65 and 75° in air and at an incident angle of75°
`in aqueous solutions after the adsorption of contaminants. The
`refractive index of the contan1inant layer was estimated to be 1.45 to
`evaluate the layer thickness. The contact angle measurements were
`performed using a KSV (Helsinki, Finland) CAM 200 contact angle
`goniometer. Both hydrophilic and hydrophobic surfaces were prepared
`
`•••
`
`for cleaning analysis. Silicon wafers were rinsed with water and ethanol
`and dried with a tlow of nitrogen and then cleaned by water plasma
`treatment at a power of 18 W for 15 min. After these treatments, the
`silicon wafers were used as hydrophilic surfaces (contact angle ~0°).
`Hydrophobic surfaces with a water contact angle of ~107° were
`prepared by modifying the silicon wafers with a hydrophobic
`monolayer. Specifically, after treatment by water plasma, the activated
`substrates were modified via vapor deposition of dodecyltrimethox(cid:173)
`ysilane at 60 °C for 6 h and then rinsed with ethanol and water and
`dried with a tlow of nitrogen to form the hydrophobic monolayer.
`
`iiili RESULTS AND DISCUSSION
`Characterization of the Electrolyzed Solutions. We
`have not directly demonstrated that the nanoparticles produced
`by electrolysis are gaseous, but the balance of the evidence is
`that they are indeed nanobubbles. Consistent with previous
`reports of nanoparticle production from solutions super(cid:173)
`saturated by electrolysis, we will henceforth refer to them as
`nanobubbles. 19-~\S The size and concentration of nanobubbles
`produced by electrolysis were determined using the N anosight
`instrument. Samples of ec-H20 NaCl were filtered through a
`450 nm filter before introduction into the NanoSight cell. The
`NanoSight revealed a population of nanobubbles of concen(cid:173)
`tration (750 ±SO) X 106 particles/mL (Figure 2). The mean
`
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`Figure 2. Histogram showing the particle concentration in a bin width
`of 1 nm obtained using the Nanosight NS300 in ec-H20 sub 450 nm,
`NaCl(aq) sub 450 nm, H 20 sub 450 nm, and ec-H20 sub 20 nm. The
`total concentration of ec-H20 sub 450 nm was (750 ± SO) X 106
`particles/mL.
`
`size of nanobubbles for the data shown was 112 ± 2 nm with a
`mode of 93 ± 4 nm. The error bars are from repeated
`measurements on the same sample. A 10-15% degree of
`variation is typically seen between samples. As a control,
`NaCl(aq) was filtered with a 450 nm filter before measurement,
`revealing a concentration of (28 ± 2) X 106 particles/mL. The
`only difference was that this sample was not passed through the
`ec-H20 electrolysis cell. Filtering ec-H20 through a 20 nm filter
`removed the nanobubbles, resulting in a concentration of
`nano particles of ( 3 ± 1) X 106 particles/mL, which is near the
`lower limit of the range of the apparatus. Similarly, filtering the
`laboratory-grade water through a 450 nm filter showed a
`concentration of nanoparticles of (3 ± l) X 106 particles/mL.
`It is also important to ascertain the lifetime or the stability of
`the nanobubbles that are produced by electrolysis. The stability
`
`c
`
`XXX-XXX
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`CONFIDENTIAL - ATTORNEYS' EYES ONLY
`
`TC00124354
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`

`

`Langrnulr
`
`A
`
`H!O
`
`! .. .. 00
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`50
`
`Particle lilize vs Time
`
`Particle size vs Concentration
`
`•••
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`B
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`100
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`
`Figure 3. (A) Mean bubble size distribution for ec-H20 sub 450 nm measured using light scattering over 10 h. (B) Histogram showing the particle
`concentration in a bin width of 1 nm measured using the Nanosight on samples of ec-H20 sub 450 nm over 5 days.
`
`of the solutions was first examined using dynamic light
`scattering (Zetasizer, Malvern). An ec-H20 sub 450 nm sample
`was checked at intervals to ascertain
`the size of the
`nanobubbles. The cuvette was left in place throughout the
`measurement. The particle size was found to be stable for 10 h,
`as shown in Figure 3a. These experiments were hampered by
`the formation of bubbles on the surface of the cuvette
`preventing the size of the nano bubbles from being followed for
`longer periods.
`To evaluate the stability over a longer period of time, a
`freshly prepared sample of ec-H20 sub 450 nm was measured
`at an interval of 1 day using the N anoSight. The syringe was
`filled with the sample
`immediately after production and
`remained undisturbed and sealed for 5 days. Each day, more
`of the sample was measured. Daily measurements confirmed
`that the size did not change significantly. Although the mean
`nanobubble size increased from 96 nm on the first day to 120
`nm on the third day, the concentration decreased significantly
`over this time, as shown in Figure 3b and Table SL This
`indicates that the size and concentration of nanobubbles are
`maintained for approximately 24 h after production.
`Note that the ec-H20 Nanoclean cell has platinum-coated
`electrodes, which degrade over time. Therefore, it is possible
`that platinum particles are being liberated as nanoparticles and
`that these nanoparticles may influence our data and/or the
`cleaning processes. A calculation was performed to determine if
`the concentration of nanoparticles observed could be due to
`platinum particles based on the known rate at which the
`electrodes degrade. This calculation revealed that if the
`nanoparticles produced were solely platinum then all of the
`platinum in the ec-H20 Nanoclean cell module would be
`exhausted after less than 30 L had been passed through the
`unit. This is inconsistent with the lifetime of the unit, which
`shows that many thousands of liters are produced before the
`platinum is substantially removed. Furthermore, our experi(cid:173)
`ments conducted over a period of more than a year (and after
`hundreds of liters have been produced) show a consistent
`production of nanoparticles in both size and concentration.
`Additionally,
`it is observed that in a given sample the
`concentration of nanoparticles observed decreases steadily
`over a period of days but the size remains rather consistent.
`This is not consistent with the presence of platinum particles,
`
`which we would expect to be either stable or grow substantially
`in size before settling in solution. We can therefore discount the
`possibility that the electrolysis process is producing a stream of
`platinum nanoparticles that are interfering in our experiments.
`At normal pH values, it has long been known that bubbles
`have a negative surface charge. This is attributed to the
`ions.32
`3 The zeta
`preferential adsorption of hydroxide
`'·'
`potential of ec-H20 sub 450 nm nanobubbles as a function
`of pH is shovvn in Figure 4. The isoelectric point (iep) is
`
`40
`
`0
`
`0 0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
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`2,5
`
`35
`
`4
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`4.5
`
`5
`
`5.5
`
`(i
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`
`7.5
`
`8
`
`pll
`
`Figure 4. Zeta potential of ec-H20 sub 450 nm as a function of pH.
`The measured zeta potential is consistent with literature reports of the
`zeta potential made on micrometer-sized bubbles. A
`
`observed at a pH of ~3.7. This is consistent with the
`measurement of the zeta potential on larger bubbles, as is the
`magnitude of the zeta potential in a solution of this
`concentration of NaCl.34
`Cleaning of Contaminated Surfaces with Nano(cid:173)
`bubbles. The first mode of cleaning that we investigated
`was whether ec-H20 could remove BSA and lysozyme from a
`hydrophilic surface. The proteins (BSA and lysozyme) were
`used as model contaminants because they had been the subject
`of earlier cleaning experiments using surface nanobubbles. i c,, i ;
`
`D
`
`XXX-XXX
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`CONFIDENTIAL - ATTORNEYS' EYES ONLY
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`TC00124355
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`

`

`Langrnulr
`
`BSA
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`Figure 5. (a) Exposure of BSA previously adsorbed on a hydrophilic silicon wafer to both nanobubble and control solutions. (b) Exposure of
`lysozyme previously adsorbed on a hydrophilic silicon wafer to both nanobubble and control solutions.
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`
`Figure 6. Adsorption inhibition study performed using ellipsometry to measure the thickness of adsorbed BSA to a hydrophilic silicon wafer as a
`function of time. Here, the thickness of adsorbed protein was measured in air. (a) BSA adsorption from NaCl solution (2.1 mM). (b) BSA
`adsorption from ec-H20 solution. (c) BSA adsorption from ec-H20 sub 20 nm solution. (d) BSA adsorption from ec-H20 sub 450 nm solution.
`
`BSA at a concentration of 1.0 mg/mL dissolved in NaCl
`solution (2.1 mM) was allowed to adsorb to a clean hydrophilic
`silicon wafer for 60 min with a wet thickness of ~2.4 nm in
`aqueous solution. Then the surface was treated with different
`types of solutions, and the thickness of the adsorbed BSA layer
`was measured as a function of time of treatment by
`ellipsometry in situ in NaCl solution (2.1 mM). To avoid the
`interference of nanobubbles in the ellipsometry measurements
`for ec-H20, ec-H20 sub 450 nm, and ec-H20 sub 20 nm
`solutions, the relevant solutions were replaced by NaCl solution
`every 10 min to measure the protein layer thickness. It was
`found that ec-H20 and ec-H20 sub 450 nm cleaned the surface
`with similar eftectiveness, whereas the control Na Cl solution
`and ec-H20 sub 20 nm solution had no eftect. (Figure~ .S). This
`fact suggests that the nanobubbles in the size range between 20
`and 450 nm contribute to the cleaning effect. This is consistent
`with the results observed in Figures 1 and 3, where the size of
`the nanobubbles
`is distributed around 90 nm. Likewise,
`lysozyme molecules were adsorbed onto the hydrophilic silicon
`wafer surface from a solution of 1.0 mg/mL lysozyme for 60
`min, producing a wet thickness of ~ 1 nm. Lysozyme can also
`be removed by ec-H20 and ec-H20 sub 450 nm solutions. In
`contrast, the NaCl solution and ec-H20 sub 20 nm solution
`
`had no cleaning effect on the lysozyme-coated silicon wafer
`surface.
`Preventing Surface Contamination. It is known that
`effective cleaning often requires mechanical agitation. This
`agitation will release contaminants from the surface; however,
`for the cleaning to be effective, it is important that the material
`is not redeposited. The suspension of soil in the cleaning fluid
`and the removal of the fluid are important in this respect. In
`situations where the cleaning process involves short periods of
`exposure to cleaning solutions of a few seconds, we considered
`the possibility that the cleaning effect of both detergents and
`nanobubbles in commercial applications may be achieved by
`stabilizing contaminants in solution that have been detached by
`mechanical scrubbing rather than removing contaminants from
`the surface. Therefore, we decided to evaluate whether ec-H20
`can prevent the deposition of material to a surface. To do so,
`we dissolved different concentrations of BSA in solutions and
`observed the adsorption of BSA to a surface over time. If the
`amount of material adsorbing to the surface is reduced, then
`this would indicate the effective prevention of deposition of the
`contaminant from solution. Figure 6 shows the adsorption of
`BSA over a 40 min period from different types of solutions. It is
`clear that the adsorption from ec-H20 and NaCl is the same at
`
`E
`
`XXX-XXX
`
`CONFIDENTIAL - ATTORNEYS' EYES ONLY
`
`TC00124356
`
`

`

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`Figure 7. Adsorption inhibition study performed using ellipsometry to measure the thickness of lysozyme adsorbed to a hydrophilic silicon wafer as a
`function of time. Here, the thickness of adsorbed protein was measured in air. (a) Lysozyme adsorption from NaCl solution (2.1 mM). (b)
`Lysozyme adsorption from ec-H20 solution. (c) Lysozyme adsorption from ec-H20 sub 20 nm solution. (d) Lysozyme adsorption from ec-H20 sub
`450 nm solution.
`
`the highest concentration of BSA studied (1.0 mg/mL) but at
`inhibits the adsorption of BSA
`lesser concentrations ec-H20
`( Pigure 6a, b).
`The electrolysis of aqueous NaCl solutions results in the
`supersaturation of hydrogen and oxygen gas but also drives a
`number of chemical reactions, including the production of
`hypochlorous acid from the production of Cl2(g) and its
`subsequent dissolution. To determine if the cleaning effects of
`ec-H20 were attributable to chemical reactions as opposed to
`nanobubbles, we investigated solutions in which the nano(cid:173)
`bubbles were removed by further filtration. We have established
`that the nanobubbles are larger than 20 nm and filtering them
`with a 20 nm filter removes them from solution (Figure 2).
`This filtration step will not remove the soluble chemical species
`produced during electrolysis, so solutions of ec-H20 sub 20 nm
`were investigated as control solutions. In this scenario, if
`cleaning is effective with ec-H20 and ec-H20 sub 450 nm and
`not effective in untreated solution or ec-H20 sub 20 nm, then
`the conclusion would be that the cleaning is due to electrolysis
`and the objects responsible for the cleaning are larger than 20
`nm and smaller than 450 nm in diameter. That is, the cleaning
`action would be attributable to nanobubbles.
`The adsorption of BSA from ec-H20 sub 20 nm and ec-H20
`sub 450 nm under the same conditions was also measured
`(Figure 6c,d). In the absence of nanobubbles (ec-H20 sub 20
`nm), the adsorption is greater, indicating that the nanobubbles
`play a direct role in preventing the adsorption of BSA to the
`surface. The adsorption from NaCl and the adsorption from ec(cid:173)
`H20 sub 20 nm are compared directly, as is the adsorption
`from ec-H20 and ec-H20 sub 450 nm (Figure Sl). vVhat is
`found is that the data does not significantly differ between the
`sub 20 nm,
`two control solutions of NaCl and ec-H20
`indicating that the chemical changes to the solution that take
`place during electrolysis have a minor effect on the adsorption
`of BSA It is also apparent that the results for ec-H20 and ec-
`
`H 20 sub 450 nm are almost identical and, as described above,
`reduce the adsorption of BSA It is therefore apparent that
`nanobubbles (not larger bubbles or chemical effects) are
`responsible for the inhibition of BSA adsorption to the surface.
`Lysozyme is a protein that we have previously found to
`adsorb strongly to surfaces and is harder to remove from a
`surface than BSA protein using surface nanobubbles. 16 It
`therefore poses a more significant cleaning challenge. The
`adsorption of lysozyme to silicon wafers in the presence of
`nanobubbles was investigated to determine if nanobubbles are
`also effective at reducing the adsorption of lysozyme. The
`results found for lysozyrne parallel those of BSA and indicate
`inhibit the adsorption of
`that the nanobubbles in ec-H20
`lysozyme to the surface (Figure~ 7). The control solutions
`showed that the chemical changes accompanying electrolysis
`were not responsible for the