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Generation and stability of bulk nanobubbles: A review
and perspective
Limin Zhou1,3, Shuo Wang2,3, Lijuan Zhang1 and Jun Hu1
Abstract
Bulk nanobubbles (BNBs) are submicron gaseous domains
dispersed in solutions, which are supposed to survive for
several hours or even days. In recent years, there has been a
rapid growth in the research and extraordinary applications of
BNBs. Conventional theories based on gas diffusion and
Laplace pressure, however, predicted that nanoscale gas
bubbles in water should dissolve within microseconds,
presenting a modern-day paradox in current nanobubbles re-
searches. Also, it is still challenging to efficiently produce
BNBs and determine their gaseous nature with the available
techniques. In this review, we start from a general introduction
and brief history of nanobubbles researches and revisit the
current progress on the generation methods and detection
techniques. Two possible formation mechanisms are sug-
gested, and the plausibility of the proposed theories on BNBs
stability is discussed with some suggestions for future studies
on bulk nanobubbles.
Addresses
1
Shanghai Synchrotron Radiation Facility, Shanghai Advanced
Research Institute, Chinese Academy of Sciences, Shanghai, 201204,
China
2
The Shenzhen International Innovation Institutes of Advanced Elec-
tronic Materials, Shenzhen Institutes of Advanced Technology, Chinese
Academy of Sciences, Shenzhen, 518055, China
Corresponding authors: Hu, Jun (hujun@sinap.ac.cn); Zhang, Lijuan
(zhanglijuan@sari.ac.cn)
3
Equal contribution.
Current Opinion in Colloid & Interface Science 2021, 53:101439
Edited by Vince Craig, Marie Pierre Krafft and Thomas Zemb
This review comes from a themed issue on Hot Topic: Nanobubbles &
Nanodroplets
For a complete overview see the Issue and the Editorial
https://doi.org/10.1016/j.cocis.2021.101439
1359-0294/© 2021 Elsevier Ltd. All rights reserved.
Keywords
Bulk nanobubbles, Stability mechanism, Generation, Detection.
Introduction
The dissolution of gas molecules in water or solutions is
a common phenomenon in nature. There is a funda-
mental question need to be answered: in what kind of
state do gas molecules exist in liquids? Conventional
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Current Opinion in
Colloid & Interface Science
concepts include single-molecule dispersion solutions
and large gas bubbles; however, it is not clear whether
the nanoscale clusters or bubbles of gas molecules would
exist. In the 1940s, it was proposed that small gas nuclei
(small gas masses) in the blood and tissues of animals
would appear at low pressures, and they could be
removed by centrifuging, filtering, boiling, and high
hydrostatic pressures and such cavitation nucleus are
theoretically stable by attaching on the sidewalls of a
hydrophobic conical crevice [1]. This suggested that
nanoscale gas bubbles might be ubiquitous in nature,
but they were not directly, observed possibly because of
the limitations of the detection techniques at that time.
In 1950, Epstein and Plesset developed a theoretical
model on the dissolution of gas bubbles in liquids based
on diffusion theory and the Laplace equation. This
theory was proved to be very successful in explaining the
dynamics of microbubbles in liquids [2]. According to
the Epstein-Plesset theory, a bubble of 100 nm in radius
will have an internal pressure about 14.4 times the at-
mospheric pressure and it cannot survive for less than
1 ms. This appears to have denied the stable existence of
nanoscale gas bubbles in liquids, and then few re-
searches were reported on this issue afterward; however,
in 1994, Parker et al. reproposed the concept of nano-
bubbles in order to offer a satisfactory explanation on
the mysterious long-range attractive forces found in
between the two hydrophobic surfaces immersed in
aqueous solutions [3]. More importantly, direct obser-
vations of surface NBs were published first by the two
groups of Hu [4] and Higashitani [5] in the 2000s, and
they were found quite stable on the surfaces in water
solutions [6e11].
According to whether NBs are attached to a surface or
dispersed in water, they are now categorized into ‘sur-
face nanobubbles (SNBs)’ and ‘bulk nanobubbles
(BNBs)’, as well as nano pancakes and multilayer
structures as reported [12,13]. Although the study of
NBs has been accompanied by controversy since the
beginning, and the paradox of a short lifetime predicted
by the Epstein-Plesset theory and the observed long
lifetime of NBs in water is yet to be resolved [14e18], it
has been recognized that the stable existence of NBs
may have significant effects on many important pro-
cesses such as protein folding, peptide self-assembly
[19], boundary slip [20], and activities of electro-
chemical reactions [21e23].
Current Opinion in Colloid & Interface Science 2021, 53:101439
2 Hot Topic: Nanobubbles & Nanodroplets
On the other hand, the applications of NBs, especially
BNBs, have not been hindered by their controversy in
the fundamental researches. In recent years, NBs have
been widely used in water treatment, aquaculture,
agricultural cultivation, health preservation, mineral
flotation, and many other fields [24e27]. The
number of published articles and citations is fast
increasing yearly. Several reviews were already published
recently to highlight the main issues regarding surface/
bulk NBs and their potential applications [9,10,24].
Due to the importance and diversity of application fields
of BNBs, this paper will give a brief review of the
research history of BNBs, their generation, and detec-
tion methods, as well as the discussions on the stability
mechanisms.
The history of BNBs
BNBs are usually defined as gas-filled domains that have
a typical diameter of less than 1000 nm in water or so-
lutions. Herein, we have listed some landmark works
concerning the studies of BNBs in history, focusing on
the experimental findings. The theoretical advances are
not included here. Figure 1 showed the historical de-
velopments of the researches on BNBs, illustrating the
main events and discoveries in the last half century.
In the early1950s, Donald A. Glaser discovered that
when charged particles passing through a superheated
liquid in a glass chamber, the liquid around the particle
trajectory would vaporize and leave a tract of microsized
bubbles to be visualized [28]. Stable micro or submicron
bubbles were found in seawater and distilled water since
the 1980s, and they were used to explain the phenom-
ena such as the large anomalies in acoustic data acquired
by navy sonar, the decompression illness among scuba
divers, and the gas nuclei in classical nucleation [29,30].
Figure 1
Some milestones of the experimental researches and developments on BNBs.
Current Opinion in Colloid & Interface Science 2021, 53:101439
The stability of those small bubbles was interpreted as
the gas diffusion that was impaired with the coverage of
impurities or surfactant on the bubble surfaces.
Due to the fast development of light scattering tech-
niques and charge-coupled devices since the 1980s, the
detection of small particles in suspensions became
possible. In 1992, it was demonstrated that clusters of
ion-stabilized submicrobubbles (bubstons) could exist
in aqueous salt systems like NaCl solutions [31,32]
despite the fact that the control experiments for veri-
fying the gaseous nature of formed submicrobubbles
were largely absent. Several other groups also claimed
that they obtained BNBs in aqueous solutions with
some methods, such as ultrasonic cavitation [33], gas
and liquid mixing, or pumping through the porous
membrane [34,35]. BNBs were also reported to be
stable in alkaline solutions while becoming unstable in
the solutions with higher ionic strength [35,36]. More
direct evidence of NBs in the water came in the 2010s;
the gaseous state of NeN stretching vibration of N2 NB
was identified by Raman spectral [37], and the spherical
structure with the radius of hundreds of nanometers was
observed on the freeze-fractured replica of NB solutions
by electron microcopies [38]. After that, more and more
papers on BNBs were published.
Generation of BNB
In this section, we will first discuss the basic principles
of NBs formation in solutions. As shown in Figure 2, two
pathways could be involved. One is the emergence of a
new phase (gas bubble phase) from the liquid phase.
This is normally a first-order phase transition and starts
from a process called nucleation, which has been well-
studied since the first description by Gibbs in the
nineteenth century. Another pathway is the collapse or
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Figure 2
Basic principles for BNB generation. (a) Classical nucleation theory for bubble formation. (b) Two possible pathways leading to the formation of stable
BNB suspensions.
shrinkage of microbubbles, where the exact mechanism
is not clear and remains controversial. We summarize the
reported methods of BNB production in recent years
and categorize them into several branches based on the
specific principles or mechanisms behind them. The
main features (gas type, size, and concentration) of the
produced BNBs and their characterization techniques
are described in Table 1.
Bubble nucleation
Bubble formation in the solution represents the forma-
tion of a new thermodynamic phase (gas) from a liquid
that is normally at a metastable state. In classical
nucleation theory, the rate of nucleation R is predicated
from the Gibbs free energy cost DG of forming the new
phase in an exponential relationshipwexpð 
DG =kB T Þ, where kB is the Boltzmann constant and T is
the temperature. In the scenario of homogeneous
nucleation, the free energy cost DG of forming a
spherical bubble nucleus in a gas oversaturated solution
would be written as:
4 3
DG ¼ pr DGlg þ 4pr 2 g
3 nescence and has been intensively studied since the 90s
where r is the radius of the nucleus, g is the surface tension
of gas/liquid interface, DGlg is the difference of Gibbs free
energy per unit volume between the bulk liquid and the gas
nuclei at the same pressure. DGlg is always negative since
nucleation normally occurs in a supersaturated condition.
Obviously, the surface free energy term of 4pr 2 g will
dominate the bulk free energy term with r 3 for a small
radius r, as in Figure 2a, resulting in a positive DG and
creating an energy barrier for the bubble nucleus to grow
homogeneously. Thus, homogeneous nucleation can be
very slow and rarely occurs, and the bubble formation in
solution is largely seen as a heterogeneous nucleation
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Generation and stability of bulk nanobubbles Zhou et al. 3
process. A certain surface or some impurities are often
required. Their presence will significantly reduce the sur-
face area of nuclei far below 4pr 2, and thus, lower the
surface free energy term withinDG, and in turn, the het-
erogeneous nucleation is more likely, and the rate of
nucleation is much faster. From the above analysis, the
main physiochemical principle for generating BNBs would
be the heterogeneous growth of bubble nuclei in a gas
oversaturated solution. The oversaturated condition can be
acquired either by dissolving/mixing more gas into the
liquid or by gas evolution chemical reactions like hydrogen
and oxygen evolution reactions.
Bubble collapse
Spherical bubbles in liquids are not stable, and they will
undergo a process of either expansion and removal
through buoyancy or shrinkage and finally collapse. The
bubble dynamics can be perfectly explained by the
famous Rayleigh-Plesset equation, except the final stage
of bubble collapse (also called implosion), which would
result in extremely high pressure and temperature
inside the bubble. Light emission is observed when
cavitation bubbles finally implode, and this amazing
(1) ‘sound to light’ phenomena is well known as sonolumi-
[55e57]; however, during the process of bubble collapse
where an NB-stage is definitely involved, there could be
another pathway for microbubbles to shrink to NBs and
become stable, as presented in Figure 2b. This idea has
been explored recently by considering the radius-
dependent surface tension at the much-curved gas/
liquid interface of NBs, implying both mechanical and
thermodynamic stability of spherical NBs in water [58].
The hydrophobic impurities attached to the gaseliquid
interface will change the surface tension, as well as the
diffusion condition at the bubble interface [14,16], and
this offers another possibility to achieve stable BNBs
Current Opinion in Colloid & Interface Science 2021, 53:101439
Current Opinion in Colloid & Interface Science 2021, 53:101439

4 Hot Topic: Nanobubbles & Nanodroplets

Table 1

Methods of generating BNBs.

Principle/Methods Chemical Number density (108/ Size Detections Methods of References

compositions mL) distribution validation
(nm)

Ethanol-water Exchange Air e4 100e200 NTA Untreated control Qiu.et al. CPC(2017) [39]
Electrolysis of 2.1 mM NaCl O2, H2 7e8 50e150 NTA Untreated control Zhu.et al. Langmuir (2016) [40]
(aq)
Mechanically mixing gas CO2 1e3 80e130 NTA ATR-FIIR spectra Seung. et al.
and liquid Langmuir (2017) [41]
Electrolysis of Na2SO4/H2O O2, H2 – <200 Differential Refractive index Alexander.et al. PLOS ONE(2017) [42]
interference contrast
Decompression at lower Air e0.4 150e250 NTA Untreated control Fang.et al. JPCC(2018) [43]
pressure
Turbulent jet flows (GO Air ~10 100e200 DLS Untreated control Marziyeh. et al. Carbon (2018) [44]
assisted)
Acoustic cavitation Air 2e9 100e120 NTA Untreated control N. Nirmalkar. et al. Langmuir (2018) [45]
Ultrasonic cavitation Air e0.7 50e150 NTA Untreated control Mo et al.
CPB(2018) [46]
Emulsion C3F8 filled e3.16 (diluted) 180e290 SEM, TEM, DLS, NTA Gas Flow Jing Du. et al.
Hollow polymeric Sci Rep (2018) [47]
Pressurized through porous- N2, O2 – 160e350 DLS, DO Dissolve oxygen Ulatowski et al. CHEM ENG PROCESS (2018) [48]
membrane
Pressurized by multiphase Air 0.5e1.5 (DI water) 150e250 NTA, Zeta potential – Ramiro et al. Mineral Eng.(2017) [49]
pump 23e41 (a  Terpineol)
Repeated compression SF6 7.6e192 e240 DLS, NTA, ATR–FTIR – Juan Jin et al. Langmuir (2019) [50]
Hydrodynamic cavitation N2,O2,CO2 – 50e450 DLS, Zeta potential, FF-TEM Untreated control Qiaozhi Wang et al. Sci Rep (2019) [51]
Hydrodynamic cavitation Air, Ar, N2 2e10 50e200 NTA, Zeta potential Degassing control Gianluca et al.Nanoscale (2020) [52]
External electric fields O2,CH4 – 220e300 DLS, DO gas solubility Mohammad et al. Sci Adv (2020) [53]
Alternative magnetic field Air e0.6 50e200 NTA, Zeta potential Untreated control Nhi Vu-Y et al. ACS AMI (2020) [54]
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(AMF)

from the microbubbles [59]. It is worth noting that the
mechanism of microbubbles shrinking to stable NBs has
not yet been well interpreted, and disputes on this hy-
pothesis remain to be resolved.
General strategies
Before introducing the specific methods, it is important
to keep in mind that the demands for efficient BNBs
production are mostly application-driven, and the vali-
dation on whether the products are gaseous bubbles or
not is sometimes left aside due to their game-changing
potentials, while it is vital to be certain on this issue
for scientific research on BNBs. Evidences on the gas-
related properties of the produce objects either strong
or weak are required to be included in any of the BNBs
research. Some of the commonly used methods for
validation of gas bubbles are listed in Table 1 and
discussed below.
1) Untreated control: Comparison between experi-
mental groups and untreated groups. After certain
treatments with gas involved, if the number density
of the observed nanosized objects did increase, this
could imply the increased ones are possibly gas-
related. Although such evidences are rather weak,
it is still very essential for scientific research to
include untreated control experiments.
2) Degassing control: Compare the experimental re-
sults before and after degassing process. After
degassing, a significant decrease in the number
density of the observed nanosized objects would
indicate their gas-sensitive property. It should note
that degassing process could also eliminate droplets
(like volatile ones), and in some cases, even nano-
particles. This is an indirect validation but important
for BNBs research especially when direct
evidence of gas bubbles is lacking.
3) Other methods: These methods are included in the
‘detection of BNBs’ part and are described later.
(i) Cavitation-pressure change
If there is gas dissolved or mixed in the liquid, the rapid
change of the pressure in a liquid, namely cavitation, will
induce the formation of gas bubbles at the positions
with lower pressure according to Henry’s law. This
principle has been widely applied to produce large
visible bubbles or microbubbles with sizes down to
10 mm. Despite the predicted short lifetime (wmsÞ of
nanoscale bubbles in water by Epstein-Plesset theory in
the 1950s [2], reports of stable BNBs suspensions pro-
duced by cavitation-based methods have been
increasing dramatically in recent years [see Table 1].
Ultrasonic cavitation and compression/decompression
through pumps are commonly used in those methods.
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Generation and stability of bulk nanobubbles Zhou et al. 5
The average bubble sizes and number densities of the
produced BNBs are around 50w400 nm and 107w109/
ml. The decompression time and the pH of the solution
are found to affect the concentration of produced BNBs
[60]. Unlike previous beliefs that nanobubble could only
appear in a gas oversaturated condition, it was proposed
that BNBs could be generated upon decompression and
stay in an unsaturated gas environment for a long time
[43]. Some studies also revealed that the bubble sizes
could be adjustable by periodic pressure change [50,51]
or pressurized through the porous membrane [48], and
the efficiency of BNBs production could be improved by
introducing some additives like graphene oxide [44],
nanoparticles [61], and organic impurities [62,63].
(ii) Gas oversaturation
Electrochemical reactions. The continuous formation of
gas bubbles is the most significant feature of the elec-
trochemical gas evolution reactions, such as oxygen
evolution reaction and hydrogen evolution reaction.
Generally, gas bubbles will first nucleate and grow on the
electrode surface before finally detach from the surface
and float. In recent years, intensive studies have been
carried out to understand the surface NBs within these
gas evolution reactions both experimentally [64e66]
and theoretically [67]. It is also reported that free
nanoscale bubbles or clouds of them were generated
during water electrolysis and could be determined
through the change in optical path or reflection index by
light scattering [42,68]. It is proposed that combustion
of H2 and O2 could happen spontaneously inside the
cloud of NBs [69,70]. Several recent studies have
characterized BNBs from electrochemical reactions and
given out the size and number density as 50w200 nm
and 7w8  108/ml [40,42].
Solvent exchange process. By displacing a gas/oil-satu-
rated solution (i.e. ethanol) with a poor solvent (i.e.
water) creates a state of oversaturation for nucleation,
the solvent exchange process has been proven to be a
convenient and effective way to form surface NBs and
nanodroplets [10]. The same idea has also been adopted
to produce BNBs [39,71e74]. According to the report,
these observed NBs-like objects would disappear upon
degassing, and their concentrations will decrease by the
time, which could imply their gaseous nature [39]. The
generation of BNBs by a similar exchange process has,
however, sparked intense debate among different
research groups with both positive and negative results
provided [39,73,75e77]. One study argued that these
objects from the solvent exchange were nanodroplets or
nanoparticles instead of gas bubbles based on their
response to external pressure and their density profile
Current Opinion in Colloid & Interface Science 2021, 53:101439
6 Hot Topic: Nanobubbles & Nanodroplets
[75]. In another study, by monitoring the whole etha-
nolwater exchange process under different replacing
timewith light-scattering technique, the authors found
out that the average size of the formed objects would
decrease down to astable value during the exchange
process, thus they suggested that these objects are likely
to be gas nanobubbles.
(iii) Compression of microbubbles
The idea of BNBs formation by the shrinkage of
microbubbles has been checked recently both from
theoretical modeling [58] and experiments [59]. The
repeated compression of microbubbles is found to be an
effective way of producing BNBs with high number
density by repeated operations [50]. Combining optical
dark-field microscopy and nanoparticle tracking tech-
nique, the dynamic process of shrinkage of micro-
bubbles composed of Xe, air, SF6, as well as the
Brownian motion of the objects left are tracked indi-
vidually [78].
(iv) Lipid encapsulation
The gas-filled lipid-encapsulated NBs as novel ultra-
sound agents can effectively enhance the contrast in
ultrasound imaging of biological targets like breast
cancer. These NBs are composed of a lipid shell (also
called armor) and a gas core containing gas C3F8 or SF6
[47]. Their productions are based on the procedure of
oil-in-water emulsion, with further gas injection and
final extraction by centrifugation. The self-assembled
lipids covering the gas core provide the stability of
these nanosized bubbles in contrast to the ‘open’ BNBs
produced by other methods [79]. Due to the same
reason, these lipid-encapsulated NBs can be dried and
characterized with high-resolution electron microscopy.
(v) External electric/magnetic field
BNBs production from external electric/magnetic fields
is quite novel and has not been noticed until recently. It
was reported that BNBs can be produced by circulating
deionized water through an alternating magnetic field,
and the consequent NB-nanoparticle interactions may
be the reason for the removal of tubercle depositions
(compose of i.e. CaCO3) on the inner wall of the pipe
from a cooling water system at the Three Mile Island
Nuclear Power Station [53]. Another study found that
by applying an external static electric field (60 V,
w12 kV/m) in a vessel containing gas and deionized
water, the gas concentration within the water increased
remarkably tow2.5 fold for oxygen and w30 fold for
methane compare to the values of Henry’s law solubility
and can be persevered for many months [54]. The
Current Opinion in Colloid & Interface Science 2021, 53:101439
reason behind this is ascribed to the massive production
of BNBs in the water, which is originated from the
enhanced surface polarization at the gasewater inter-
face by the external electric field, and thus, leads to the
cavitation at the liquid interface.
Detection of BNBs
The long-term existence of BNBs suspensions directly
contradicts the prediction of short-lived nanoscale
bubbles in water by Epstein-Plesset theory. To resolve
this paradox, it is vital to develop reliable detection
methods for BNBs in liquids. With the size of several
nanometers to hundreds of nanometers, BNBs in liquids
are largely transparent, and the direct visualization by
optical microscope has always been a difficult task.
Another challenge is how to distinguish the gas NBs in
water from other colloidal dispersions containing solid
nanoparticles, oil nanodroplets, or biological vesicles.
This is more crucial when considering the controversial
nature of gas NBs on their existence and stability.
Techniques like light scattering methods and high-
resolution electron microscopies normally have poor
chemical sensitivity，while most of the current spectral
techniques lack enough spatial resolution (  100nm) to
track and probe each individual NB. The development
of techniques with both high spatial resolution and
chemical sensitivity is an urgent need for BNBs
research.
In the following part, we will discuss the techniques
applied in the studies of BNBs in solutions, including
their advantages, disadvantages and give some sugges-
tions for future studies.
Light scattering techniques
Under the assumption that BNBs do not float quickly
and move in liquids following Brownian motion, thus
they can be detected with laser scattering-based
methods due to the high refractive index contrast be-
tween liquids and gas bubbles. A simple and convenient
way to discriminate between BNBs suspensions and
clean solutions without any bubbles or particles inside
(both are transparent in bare eyes) is by using a laser
beam to illuminate them; because of the Tyndall effect,
the laser beam is scattered and can be viewed with bare
eyes in BNBs suspensions while nothing can be seen in
the clean solution.
Dynamic light scattering (DLS)
The dynamic light scattering is based on the decays of
the autocorrelation function of scattered light intensity.
Smaller particles have a faster decorrelation of the
scattered intensity compared to the larger ones, and
thus, they can be used for size determination. While for
poly-dispersed samples, extracting the accurate infor-
mation of particle sizes from the sum of the autocorre-
lation functions has long been a difficult task, especially
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with larger particle sizes and high refractive index
contrast where multiple scattering might be involved.
Therefore, commercial DLS instruments have the
sample requirement like mono-dispersed systems with
sufficiently high concentrations to ensure the data
quality. The acquired DLS data, like the size distribu-
tion, is only the average value from the fit of autocor-
relation functions. Considering that BNBs suspensions
may often contain bubbles with different sizes and the
refractive index of the gas/bubble interface is very high,
lack of the accurate information will cause more
confusion in further data analysis. Another issue is that
DLS alone does not provide chemical information to
distinguish gas NBs from particles or droplets.
Nanoparticle tracking analysis (NTA)
NTA technique captures the movement of each scat-
tering object in the solution with a dark field microscopy
and analyzes their trajectories to deprive their sizes
respectively according to StokeseEinstein relationship
of Brownian motion:
DR2 ðtÞ kB T
¼D ¼ (2)
4t 6phr
where DR2 ðtÞ ¼ jr1 ðtÞ  r1 ð0Þj2 is the mean square
displacement of the particle.
The NTA technique is much more accurate and reliable
for size determination with a typical reliable particle size
around 30w1000 nm. As for the concentration, it should
be noted that the fluid channel for the NTA experiment
is usually very narrow (w10 mm of depth) to allow the
efficient illumination for the data acquisition by the
camera. Only an extremely small volume of liquid
(100 mm  80 mm  10 mmÞ is measured in each
image, and thus, the final concentration (or number
density) of the captured particles is obtained by multi-
plying a factor of 1:25  107 /mL. The perfect detectable
range of concentration for nanoparticle tracking analysis
is about 107 w1010 /mL, and good quality of data
normally comes in the range of 108 w109 /mL with
8w100 objects in each frame of the captured video.
Data acquisition by NTA experiments is also sensitive to
camera and image analysis settings, and a poor focus will
leave many particles untracked, and particles will drift in
an unsteady flow condition. Therefore, protocols for
NTA experiments of BNBs should be carefully
designed, and some suggestions include: capture in a
steady laminar flow, monitor for a longer time, and
repeat the measurement of both control and sample
group multiple times.
The fatal disadvantage of the light scattering-based
techniques is that they cannot distinguish gas NBs from
nanoparticles or nanodroplets. This could be the part of
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Generation and stability of bulk nanobubbles Zhou et al. 7
the reasons why some conflict results are reported on
the same process like ethanol-water exchange from
different groups [39,71,75,76,80].
Digital holographic microscopy
Unlike conventional optical microscopies record only
the intensity of light reflected or transmitted by an
object, holographic microscopy can record the whole
wavefront containing both amplitude and phase infor-
mation with two coherent beams. Their interference
pattern can then be used to recover the 3D structure, as
well as the phase image of the sample with computer
algorithms [81]. The dependence of the phase contrast
on the particle size and the refractive index enables the
holographic microscopy to differentiate different kinds
of nanoparticles in suspensions from their refractive
index. A recent study demonstrated that NTA in an off-
axis digital holographic microscope could effectively
distinguish nanoparticles from gas NBs with their
distant phase shift (bubbles are negative while particles
are positive) [82]. The holographic particle character-
ization method is capable of processing thousands of
particles and bubbles with both their sizes and refractive
index determined and thus can be a promising and
powerful tool in the future study of BNBs.
Resonant Mass Measurement (RMM)
RMM is a technique that detects particle buoyant mass
by measuring the change in frequency with its unique
microelectromechanical systems resonator and has
recently been explored as an innovative tool for char-
acterizing BNBs [83]. When liquids flow through the
microcantilever tip immersed in a suspended micro-
channel, the mass change due to the particle inside will
translate into the shifts in the resonance frequency. By
knowing the liquid density and a predefined flow ve-
locity, Resonant Mass Measurement offers the possi-
bility to probe both the gas density and the
concentration of BNBs suspensions. Studies of BNBs or
shell-stabilized NBs with RMM technique demon-
strated its capability to distinguish buoyant particles
(bubbles) from nonbuoyant particles (solids)
[79,84,85], while on the other side several studies have
attributed the observed NB-like objects from the sol-
vent-exchange process and commercial NB generators
to nanodroplets or nanoparticles which have a density
very close or higher than that of suspension liquid
[75,86].
High-resolution imaging techniques
Electron Microscopy (EM) technologies like Trans-
mission Electron Microscopy (TEM) and Scanning
Electron Microscopy (SEM) are applicable only to
shell-stabilized NBs since they require the sample to
be dried and then covered with depositions to gain
enough contrast. To enable imaging liquid samples with
techniques such as liquid-TEM or Electron Energy
Current Opinion in Colloid & Interface Science 2021, 53:101439
8 Hot Topic: Nanobubbles & Nanodroplets
Loss Spectroscopy, a thin and closed liquid cell-like
Si3N4 window is required, and by that, the mobility of
NBs is severely restrained, and thus, they should be
regarded as surface NBs [87,88]. Cyro-EM or Freeze-
fracture electron microscopy can preserve the original
structure of the sample by rapid freezing and have been
demonstrated as the promising tools in the study of
BNBs suspensions owing to the high-resolution and
capability of providing direct evidences among bubbles,
solids, and droplets [37,38,89]. The effects of cryofix-
ation, fracture and replication upon the sample struc-
ture should, however, be carefully evaluated. It is
reported that NBs in suspensions may agglomerate or
coalesce to larger bubbles after the freezing process
[80].
Spectral techniques
Spectroscopies contain chemical information, which is
crucial to probe the gas composition and gas state of
BNBs in suspensions. Examples of spectral data from
BNBs solutions include Fourier Transform infrared
spectroscopy of CO2 [41], Raman spectroscopy of N2
[37], near-edge X-ray absorption spectroscopy of O2
[90], X-ray fluorescence spectroscopy of Kr and SF6
[60], and small-angle neutron scattering of water vapor
[91].
The main issues among BNBs researches with tradi-
tional spectral techniques come from the two disad-
vantages: the lack of enough spatial resolution (several
hundred nanometers at best) to probe single NB and the
signal may come from the multiple bubbles, as well as
the gas molecules dispersed in the liquids; the strong
absorption by water in a wide spectrum range, which
makes it difficult to acquire data with a high signal-to-
noise ratio, and thus, complicates the spectral analysis.
Fortunately, synchrotron radiation-based spectroscopies
like X-ray absorption spectroscopy, X-ray emission
spectroscopy, and resonant inelastic soft X-ray scat-
tering, which probe the chemical or electronic structure
of the specimen, could provide element-sensitive in-
formation with both high spatial resolution and photon
energy resolution. Recent developments of these tech-
niques have enabled the capability to conduct in-situ/
operando experiments in a liquid or biological system,
which can be a promising tool to reveal the intriguing
properties of BNBs.
Zeta potential measurement
The measured zeta potentials in many of the
literature about BNB often have a negative value ranging
from 50mV to  20mV. This led to the preconceived
idea of negative charged BNBs in suspensions which are
increasingly popular these days. The scenario of nega-
tive charged BNBs provides explanations for NB
Current Opinion in Colloid & Interface Science 2021, 53:101439
stability as follows: the strong electrostatic repulsion of
net electrical charge can resist processes like coales-
cence into large bubbles and Oswald ripening, which
both are devastating for stable dispersions of BNBs.
The basic principle of technique for zeta potential
measurements is briefly introduced as follow: when
applying an external electric field E across the sample
solution, the movement of particles will result in a small
frequency shift Df which is related to the particle
velocity vp by:
 
q
Df ¼ 2vp sin l (3)
2
where q is the scattering angle and l is the wavelength of
the scattering beam. Some other techniques determine the
phase change Df $t with time t instead of frequency shift to
improve the performance. The electrophoretic mobility of
dispersed particles me ¼ vp =E then can be converted to
zeta potential Z with the relationship of:
2ε
me ¼ Z$ f ðk $ aÞ (4)
3h
where ε and h is respectively the dielectric constant and
the absolute zero-shear viscosity of the medium, f ðk $aÞ is
the Henry’s equation with 1=k the Debye length and a the
radius of a spherical particle. f ðk $aÞ has the minimum
value of approximately 1.0 in nonpolar media while the
maximum value of 1.5 in polar media.
Notice that the shift in frequency or phase of the
scattering light is associated to each particle inside the
solution, while in the air NB-water system, the re-
sponses of the substances to an external electric field are
most likely to be H3 Oþ , OH , HCO 2
3 , CO3 or some
charged impurities. The momentum of exchanges from
these ionic species under electric field could change the
velocities of BNBs even when their surfaces are not
actually charged [58]. Another study of BNBs produc-
tion via external electric fields proposed that the in-
teractions between surface dipoles, which are arisen
from the polarization at the water surface of NBs, can
provide sufficient repulsive force to counter Laplace
pressure and thus stabilize the NBs without the need for
accumulation of surface charge at the bubble surface
[54]. These have raised strong suspicions on the validity
of the ‘negative charged BNBs scenario’.
The measured zeta potentials are also highly dependent
on the pH and salt concentrations of the solutions, and
also the electrical potentials do not tell whether they are
from the gas/liquid interface or solid/liquid interface;
therefore, the usage of the measured zeta potentials as
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an indicator for the surface charge properties of BNBs, as
well as their stable mechanism should be reconsidered
and carefully evaluated.
As discussed above, all the current techniques do have
some sorts of disadvantages on the exact and effective
detection of BNBs in water. It is still challenging to
distinguish gas NBs from nanoparticles or nanodroplets,
both with enough spatial resolution (w20 nm) and
chemical sensitivity. To avoid confusions in the future
study, some advice for the BNB experiments are listed
below:
(1) Untreated Control groups and degassing control
should be conducted to check if the BNBs are
actually gas-related;
(2) The purity of the gases and liquids involved should
be as high as possible; use clean vessels, tubes, and
containers in each step, such as production, trans-
ferring, and storage. Glass, Teflon, or stainless steel
are recommended. Mechanical parts like pumps
should avoid oils.
(3) Any intentional or unintentional introduction of
impurities like organic compounds should be avoi-
ded. If they are indeed necessary, their properties in
gas-free liquids, such as accumulation and agglom-
eration, should be addressed. There is a strong
possibility that such a process will lead to the
introduction of nonbubble objects instead of gas
NBs.
(4) More direct evidence should be provided to validate
that the studied BNBs are indeed gaseous. The
measurements of density, compressibility, refractive
index, and certain spectra related to gas-phase are
strongly recommended.
Stability of BNBs
Although the stability mechanism of NBs is not clear,
various theoretical attempts have been made to resolve
the problem. In this section, we first introduce a stan-
dard thermodynamic analysis of the stability of indi-
vidual BNB, and some of the recent theories will be
reviewed, then give a brief introduction of the collective
kinetics of BNBs in a suspension. Finally, we will review
some attempts to explain the stability beyond the
classical gas theory.
Thermodynamics
Considering a spherical NB suspends in a gas dissolved
aqueous solution in ambient pressure P0 and tempera-
ture T, the pressure inside the NB is P ¼ P0 þ DP,
where the Laplace pressure is given by, DP ¼ 2g= R,
where g is the surface tension of the gas water interface
and R is the radius of the bubble. The dissolution
equilibrium between gas inside the bubble and solution
is given by Henry’s law, H = c/P, where H is the Henry
constant of the gas and c is the gas concentration in the
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Generation and stability of bulk nanobubbles Zhou et al. 9
solution. The chemical potential difference between
the gas inside the bubble and the surrounding liquids is
given by,
Dm ¼ kB TlnðPH = cÞ (5)
which controls the transport of the gas between the
bubbles and the surrounding solutions, where kB is the
Boltzmann constant. If Dm < 0, then the gas inside the
bubble will flow into the surrounding liquid, and vice
versa for Dm > 0 the gas in the surrounding liquid will
flow into the bubble. By signing Dm = 0, we get a critical
radius,
2g
Rcritical ¼ (6)
c=H  P0
A bubble equilibrates with surrounding solution when
R ¼ Rcritical . Rcritical only exists when c/H > P0, that is,
when the solution is supersaturated, providing that the
g is positive. It should be noted that this equilibrium is
not stable, since,
vðDmÞ 2kB T g
¼  <0 (7)
vR PR2
A perturbation of the bubble radius will lead the bubble
to dissolve in solution or grow indefinitely.
The above analysis showed that the BNBs are thermo-
dynamically unstable. To achieve a stable bulk nano-
bubble, a necessary condition is vðDmÞ/vR > 0.
Combining with Eq. (5) one gets,
d ðDPÞ
>0 (8)
dR
which is a key result for understanding the stability of
BNBs. It implies that if the Laplace pressure radius curve
shows a positive slope, then a stable BNB can be expected.
In the above analysis, we have assumed the surface tension
of the gas liquid interface is a constant, then d (DP)/dR is
definitely negative. If the surface tension is size-dependent
g(R), then Eq. (8) may be satisfied.
There are several mechanisms that can lead to the
radius-dependent surface tension of bubbles. For
example, when the size of a bubble is comparable with
the scale of the intermolecular forces, the surface ten-
sion is intrinsically size-dependent, which is also known
as the Tolman effect; however, this effect is significant
only when the bubble size is in the scale of 1 nm,
whereas, the experimentally observed nanobubbles are
usually in the scale of 100 nm. Another possible mech-
anism is caused by the adsorption of trace amounts of
Current Opinion in Colloid & Interface Science 2021, 53:101439
10 Hot Topic: Nanobubbles & Nanodroplets
contaminants in the water, which may be trapped at the
interface due to their hydrophobicity. When the bubble
dissolves, the surface density of the contaminants in-
creases and leads to the reduction of the interfacial area,
which results in a size-dependent surface tension of
bulk nanobubbles. This mechanism was first proposed
by William Ducker to explain the stability of surface
nanobubbles in 2010 [92] and was recently revised by
many researchers to explain the stability of bulk nano-
bubbles. Since those surfactants are insoluble in water,
after the adsorption they pin on the interface. The
reduction in the area of gas/water interface would
reduce the surface tension, as described by many studies
on Langmuir films. In the high compression state, Eq.
(8) may be satisfied, and then a stable BNB could be
achieved. In fact, it is well known that the bubble sta-
bility can be significantly increased by the adsorption of
insoluble surfactants at their interface, which has been
widely used for producing stable microbubbles for an
ultrasound contrast agent. The contaminants in the
solutions may have many origins, but the characteristics
of the surface pressure-area isotherms are similar. If we
use the insoluble surfactant Dipalmitoyl Phosphatidyl-
choline as a model surfactant for analysis, when the
surface concentration of the surfactant reaches 2.5/nm2,
a minimum surface tension of about 3 mN/m is
achieved. If the number density of bulk nanobubbles is
about 108/ml, the total surfactant adsorbed on bubbles is
in the order of 107 M. Due to the nonzero surface
tension, supersaturation is required to balance the
chemical potential difference between inside and
outside the bubble, and this supersaturation may occur
during the bulk nanobubble generation process.
Above, we have qualitatively analyzed the influence of
supersaturation and adsorbed contaminants on the sta-
bility of bulk nanobubbles. Of course, this mechanism
needs more quantitative research, including the rela-
tionship between the number density of the nano-
bubbles and the generation methods, the type of the
contaminants and their concentration, etc.
Recently, Tan et al. [18] proposed that BNBs can be
stabilized by the charges pinned on the gas/water
interface, in this case, the effective Laplace pressure is,
2g Q2
DP ¼  (9)
R 32p2 R4 ε
where Q is the total charged adsorbed on the interface, ε is
the dielectric coefficient of the liquid. The second term on
the right-hand side of Eq. (11) is the electrostatic stress
caused by the charges pinned on the interface. Inserting
this into the equation, one gets,
Current Opinion in Colloid & Interface Science 2021, 53:101439
Q2
R3 < (10)
16p2 gε
which is a constraint on the bubble radius. The authors
obtained the charges pinned on the bubble by analyzing
the zeta potential data of the micron-sized bubbles, the
stability under different gas saturation conditions is also
provided. While one should note that, as discussed in the
‘zeta potential’ part, the measured negative values of BNBs
do not actually mean that the bubble surface is negatively
charged, and the frequency shift under an external electric
field may originate from the movement of ions or charged
impurities already existing in the solution instead of indi-
vidual nanobubbles. On the other hand, considering the
shrinkage of a negatively charged nanobubble, the charge
diffusion at the air/water interface with a typical length
scale l of 1w2:5 Å is too fast (l 2 =Dw1010 s) than the
bubble shrinkage itself (w1msÞ and the measurement of
zeta potential (w10ms), the significant decrease of zeta
potential with smaller bubble radius seems
quite confusing. Furthermore,the mechanisms of pinned
charges and pinned contaminants can be considered as two
ways of achieving size-dependent surface tension. When
the density of the adsorbents at the gas-liquid interface is
high enough, the repulsion between these adsorbents will
always leads to a stable bulk nanobubble, regardless of the
specific forms of the interaction between them is electro-
static or steric.
Kinetics
When BNBs are in an unstable state, we expect to
further understand the time they take to completely
dissolve or grow into the macroscopic bubbles. The ki-
netic problem of the gas transport between a bubble and
the surrounding liquid was first solved by Epstein and
Plesset [2]. In this section, we first generalize the
Epstein-Plesset model to include the size-dependent
surface tension. We then give a brief introduction of
the diffusional interaction between BNBs.
Considering a BNB with radius RðtÞ locates at the origin
and immerses in a large fluid domain. The gas concen-
tration far away from the bubble is c0 f ; where c0 the
solubility of the gas at 1 bar, and f is the saturation level
of the liquid. The gas concentration at the fluid inter-
face is determined by Henry’s law,
cðRþ ; tÞ ¼ HðP0 þ DPÞ (11)
where DP is the Laplace pressure.
The concentration field in the fluid domain is governed
by the steady-state diffusion equation, Dc ¼ 0. The
solution of this equation in the spherical coordinates is,
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 Generation and stability of bulk nanobubbles Zhou et al. 11

DLS or NTA methods [52,78,89]. We believe that the

R½HðP0 þ DPÞ  cN  combination of the collective dynamics and some spe-
cðr; tÞ ¼ þ c0 f ðtÞ (12)
r cific properties of individual nanobubbles (such as partly
covered by insoluble surfactant) can be helpful to un-
derstand the formation and evolution kinetics of BNB
The flux at gas/water interface is given by, suspension.

D½HðP0 þ DPÞ  c0 f 
Jðr ¼ Rþ Þ ¼  (13)
R as an ideal gas that equilibrates with the surrounding
The current across the total gas/water interface is,
I derstand the stability out of classical gas theory. Letel-
I ¼ Jds ¼  4pRD½HðP0 þ DPÞ  c0 f  (14)
bubble surface
where D is the diffusion coefficient of the gas. The mass
conservation is given by,
    NBs. Manning proposed that there is no dissolution
d 4pR3 r 3 d 4pR3 ðP0 þ DPÞ
I ¼  ¼  (15)
dt dt 3BT
where B is the gas constant. Inserting DP ¼ 2gðRÞ=R to
the above equation yielding,
dR DH  1  f þ 2gðRÞ=RP0
 ¼    (16)
dt R state equation and Henry’s law may not be applied.
1 þ 4 3RP0 ddRg R2 þ g
where H* is the dimensionless Henry constant and is equal
to the ratio of the gas concentration in the liquid phase to
that in the gas phase. Eq. (16) describes the change of the
radius of an isolated BNB with size-dependent surface
tension.
For incorporating the diffusional interaction between
bubbles, a direct way is to specify the boundary condi-
tions (Eq. (14)) on each bubble, and the steady-state
diffusion equation can be solved numerically on the
fluid domain. The mass conservation equation for each
bubble couples to the flux at the gas/fluid interface (Eq.
(15)), then the dynamics of each bubble can be
computed. Zhu et al. applied a similar method to
calculate the evolutions of surface NB arrays [93].
Due to the low number density of the BNBs, the dis-
tance between bubbles is much larger than the size of
BNBs, which enables us to solve the kinetics using a
mean field approximation. This method was first used to
compute the Ostwald ripening process [94]. The key
idea is to use an effective gas concentration to represent
the influence of neighboring bubbles in the suspension.
It should be noted that the bulk nanobubbles suspen-
sion is always in a dynamical state, where the gas mol-
ecules transfer between the bubbles leading to the size
distribution function evolving over time, as measured by
In a previous analysis, the gas inside the NBs is treated
liquid very fast; it is very hard to explain why NBs are
tolerant to environmental changes, such as, temperature
and gas saturation. Recently, a few studies try to un-
lier and Turmine [95] gave a description of BNBs
suspension by nonextensive thermodynamics. A similar
idea was also proposed by Manning; according to the
Raman spectral of the BNB suspension, almost no ni-
trogen molecules are dissolved in the aqueous solution,
and the vast majority are present in the form of nitrogen
equilibrium between the gas inside BNBs and in the
surrounding liquids [58]. Recent scanning transmission
X-ray microscopy studies have shown that the gas inside
the NBs is in a highly condensed state, and the density
inside NBs was estimated as one to two orders of
magnitude larger than the ambient air [90]. If this
condensed gas state exists in the BNBs, then the gas
Although it is a new way to solve the stability problem of
BNBs, the relevant mechanism still needs further
exploration. There is still a long way to go before we can
have a satisfactory theory on thesurprising stability of
BNBs, especially when there are still many questions in
whether the measured objects are actually gaseous
bubbles or not. Future improvement on the effective
production and accurate detection of BNBs may pave
way for better theoretical understanding.
The prospective of BNBs
In summary, although a lot of excellent results and
progresses in the investigation of BNBs have been
published in the last twenty years, it is still in its infancy,
and many challenges remain. First, how to produce
controllable BNBs with certain small sizes (such as
below 100 nm) and higher concentrations (larger than
108/ml) is the main challenge for their applications.
Second, the current detection of BNBs mainly depends
on DLS or NTA techniques, which are limited by the
concentration range (above 109/ml for DLS) and the
resolution range (larger than 50 nm for NTA). Particu-
larly, these techniques could not tell the chemical in-
formation of the detected particles. It is very necessary
to develop new techniques with both higher spatial
resolution and sensitive chemical resolutions. Synchro-
tron radiation-based fluorescence combing absorption
may be one of the choices. Third, although several
models (charges, contamination, etc.) were proposed on

www.sciencedirect.com Current Opinion in Colloid & Interface Science 2021, 53:101439

12 Hot Topic: Nanobubbles & Nanodroplets
the mechanism of the stability of BNBs, there is still a
lack of a satisfactory picture to explain their long life-
time. Finally, it is believed that the applications of BNBs
would have more promising prospects in a wide field.
Among those researches, BNBs biological effects could
not be neglected, especially in the medical diagnosis and
the improvement of sediment. It is also worth noting
that if the high-density state generally existed in BNBs,
a lot of important applications could be expected, e.g.,
high gas density NBs may largely enhance the efficiency
of oxygen delivery and hydrogen storage, be used as
micro-nano reactors, and have unique physiological ef-
fects, etc.
Funding
This review was supported by the National Natural
Science Foundation of China (Nos. 11874379,
U1532260, 12005284), the Key Research Program of
Frontier Sciences，CAS (No. QYZDJ-SSW-SLH019)
and China Postdoctoral Science Foundation (Grant No.
2020M672765).
Author contributions
All authors contributed to the structuring and the
writing of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in this
paper.
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