All-aqueous emulsions as miniaturized chemical reactors in

All-aqueous emulsions as miniaturized chemicalreactors in the food and bioprocess technologyAshkan Madadlou1, Vittorio Saggiomo2, Karin Schroen3,4 andVincenzo Fogliano1

Available online at www.sciencedirect.com

ScienceDirect

All-aqueous emulsions are conventionally formed at bulk

scale by mild shaking of aqueous two-phase systems. They

can be used to carry out reactions both in droplets

(compartmentalized) and on droplet surfaces in conditions

free of synthetic surfactants and organic solvents. The use of

all-aqueous emulsions for extractive bioconversion is a

routine application; however, these emulsions hold many

more promises. A renowned, rapidly evolving application is

bio-microgel synthesis through biopolymer crosslinking within

the emulsion internal phase. When polyelectrolyte

crosslinking is achieved at the interface rather than in

droplets, microcapsules can be formed, and when in situ

colloidal particle generation at the droplet surface is obtained,

colloidosomes are produced. The use of microfluidics to

control the formation of all-aqueous emulsions offers many

advantages in reactions monitoring and partitioning of

reactants.

Addresses1 Food Quality and Design Group, Department of Agrotechnology and

Food Sciences, Wageningen University and Research, Wageningen, P.

O. Box 17, 6700 AA Wageningen, The Netherlands

2 Laboratory of BioNanoTechnology, Wageningen University and

Research, P.O. Box 8038, 6700 EK Wageningen, The Netherlands3 Food Process Engineering Group, Department of Agrotechnology and

Food Sciences, Wageningen University and Research, Bornse

Weilanden 9, 6708 WG Wageningen, The Netherlands4Membrane Processes for Food, University of Twente, Drienerlolaan 5,

7522 NB Enschede, The Netherlands

Corresponding author: Fogliano, Vincenzo ([email protected])

Current Opinion in Food Science 2020, 33:165–172

This review comes from a themed issue on Food physics and

materials science

Edited by Maria Teresa Pedrosa Silva Clerici

For a complete overview see the Issue and the Editorial

Available online 5th July 2020

https://doi.org/10.1016/j.cofs.2020.06.005

2214-7993/ã 2020 The Authors. Published by Elsevier Ltd. This is an

open access article under the CC BY license (http://creativecommons.

org/licenses/by/4.0/).

Conventional emulsion microreactorsMiniaturized chemical reactors provide inimitable advan-

tages over running reactions at large scales, including

reduced use of chemicals and solvents, increased safety,

enhanced reaction rates, and the ability to screen and vary

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reaction conditions pretty much at will [1]. Various sorts of

microreactors such as liquid marbles and liquid biphasic

systems (LBSs, liquid-liquid systems) have been devel-

oped and exploited. Oil-water LBSs can be effectively

miniaturized by standard emulsification (bulk scale), and

within fluidic microchannels which allows unprecedented

control over droplet size. Running reactions in oil-water

LBS microreactors enables combining immiscible part-

ners of a reaction at the oil-water interface [2,3]. Alterna-

tively, the interface might serve as the purging site for

removal of specific compounds produced in either of the

emulsion phases into the opposite phase. For example,

oil-in-water emulsion droplets stabilized by lipase-loaded

mesoporous carbon nanospheres functioned as a micro-

reactor with reactants in the oil phase for enzymatic

transesterification of phytosterols and concurrent removal

of the produced H2O across the oil-water interface into

the aqueous phase [4]. Likewise, the aqueous phase of

water-in-oil emulsions can be used as compartmentalized

microreactors, for instance, for synthesis of protein [5],

peptide [6], and starch [7] microgel particles by enzy-

matic, chemical and thermal crosslinking. Although in

limited cases, for example, during hydrolysis of vegetable

oils, surface active products can be formed, synthetic

surfactants are very commonly required to decrease the

interfacial tension at the oil-water interface, and thus

facilitate emulsification. However, these surfactants cause

high carbon footprints during their production, and are

detrimental to human health and the environment. To

mitigate this, W/O Pickering emulsions that are free of

surfactants [2] have been used; however Pickering particles

are not necessarily intrinsically safe, and they may be

cumbersome to prepare. Besides, tedious purification steps

that involve the use of organic solvents that subsequently

have to be fully removed are needed. Moreover, oil oxida-

tion of may occur during production, potentially jeopardiz-

ing quality and safety of the final product.

All-aqueous emulsions: a green alternativeAll-aqueous emulsions are genuine alternatives to water-oil

emulsions and can be used for chemical reactions without

the need of synthetic surfactants and organic phase. These

emulsions are aqueous two-phase systems (ATPSs) and

comprise two immiscible aqueous solutions. The interfa-

cial tension between the immiscible aqueous phases is

several orders of magnitude lower than at typical oil-water

interfaces [8]. This allows formation of microdroplets by

mild mechanical forces, that is, shaking/stirring at bulk


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166 Food physics and materials science

scale and perturbation of water-water (W-W) jets in micro-

fluidic channels, yielding all-aqueous emulsions [9�]. The

thickness of the W-W interface typically exceeds the

effective length of the constituent polymers and the ultra-

low interfacial tension basically takesaway thedriving force

for low molecular weight surfactants to adsorb at the inter-

face. Comparable to the oil-water interface, particles can

accumulate at the W-W interface, which hinders spinodal

decomposition and macroscopic phase separation of all-

aqueous emulsions [10].

Components that can form all-aqueous emulsions are

pairs of i) noncharged polymers for example, polyethyl-

ene glycol (PEG)-dextran (Dex), ii) a noncharged poly-

mer (e.g. PEG) with a salt (e.g. K2HPO4) or with a

charged polymer (e.g. chitosan), or iii) two co-charged

polymers [11]. Dex is listed generally recognized as safe

(GRAS) and PEG is a biocompatible polymer approved

for human oral applications [12]. Within the food sector

good quality ATPS were obtained using whey proteins

which were hydrophobized through a food-grade acetyla-

tion procedure and heat treatment [13], resulting in

immiscibility of the negatively charged proteins with

Na-alginate [14]. Based on interfacial rheology assess-

ments, it was found that the most hydrophobic particles

accumulated at the W-W interface [15], and other parti-

cles remained in the protein phase.

Partitioning in all-aqueous emulsionsAll-aqueous emulsions segregate guest molecules (such as

DNA, proteins andenzymes) through partitioning between

the two aqueous phases, as well as at the interface. Efficient

partitioning of reactants between the two (inner and exte-

rior) phases of emulsions is a prerequisite for accomplishing

chemical reactions either in the inner phase or at the W-W

interface. Partitioning in the inner phase enables running

compartmentalized reactions, whereas partitioning at the

interface allows to carry out on-droplet reactions. Within

Table 1

Examples of the bio-compounds segregated using ATPSs

ATPS pair Segregated compound

UCON‒KH2PO4 Laccase

PEG‒Na2SO4 Laccase

PEG‒Na3PO4 Recombinant chymosin

PEG‒K3PO4 Gallic acid, Epicatechin, Chlorogenic

PEG113-b-PNIPAM149‒Salt Bromelain

PEG‒Dex Immunoglobulin monomers

Immunoglobulin aggregates

Ethanol‒K2HPO4 Vanillin

Ascorbic acid

Kp: Partition coefficient.

RE: Recovery extent (%).

UCON 50-HB-5100: A random copolymer of ethylene oxide and propylene

PEG113-b-PNIPAM149: Block copolymer of PEG (mean degree of polymeriz

LCST 28.6�C.


the most common pair, that is, PEG-Dex, PEG forms the

less polar phase [11] and it can be removed by gel perme-

ation chromatography [16] once the reaction is accom-

plished. The utilization of thermo-separating polymers

such as UCON (a copolymer of ethylene oxide and pro-

pyleneoxide) facilitatespolymer recoverybyheatingabove

its lower critical solution temperature (LCST) [17]. Com-

parably, certain food-grade polymers, for example, hydro-

xypropyl cellulose have an LCST of 43ᵒC at 0 M NaCl [18].

A plethora of enzyme and other biomolecules have been

segregated/compartmentalized using ATPSs. In Table 1,

examples of ATPSs with the segregated compounds are

provided. Recovery of proteolytic enzymes from micro-

bial fermentations into the PEG-rich phases of PEG/

phosphate and PEG/Citrate is also known [19]. The

protease partitioning efficacy is dependent on PEG

molecular weight, pH, and NaCl concentration [20].

PEG retains a higher enzymatic activity of the recovered

recombinant chymosin, compared to other affinity phases

for example, poly(ethylene glycol)-block-poly-(propyl-

ene glycol)-block-poly-(ethylene glycol) [21].

Compartmentalized reactionsExtractive bioconversion using all-aqueous emulsion

reactors

All-aqueous emulsions are widely used for extractive

bioconversion. To this end, a pair of phase-forming poly-

mers (without being stabilized by Pickering particles) are

mildly stirred typically for hours to days, and consecu-

tively separated [22]. Extractive bioconversion relies on

preferential partitioning of the biocatalyst (microbial

cells, enzymes) and its substrate in one of the aqueous

phases (usually droplets) and recovery of the bioconver-

sion product(s) into the other phase. Product partitioning

away from the biocatalyst can for example, lift product

inhibition, and drive the reaction to higher conversion

rates, as well as prevent cell toxicity [11,23��].

Host phase Kp; RE Ref.

KH2PO4 >3 [17]

Na2SO4 �100% [51]

PEG 4�6 [21]

acid PEG 1.01�4.6,

>75%

[52]

PEG113-b-PNIPAM149 �95% [53]

Either PEG or Dex ‒[16]Interface �100%

Ethanol �96%[54]K2HPO4 92‒2%

oxide.

ation: 113, Mn = 5.0 KDa) and poly (N-isopropylacrylamide) (PNIPAM);


All-aqueous emulsion microreactors Madadlou et al. 167

Product partitioning into the recovering phase can be

improved by adding-specific ligands coupled to a phase-

forming polymer and engineering of the product, for

example via tagging the recombinant proteins [22]. Fer-

mentative production of pullulan in PEG-salt and PEG-

Dex ATPSs and recovery of pullulan into the PEG phase

[24] is a recent illustrative example of what all-aqueous

emulsions can deliver to extractive bioconversion.

In the food sector, ATPSs have been used for the post-

hydrolysis fractionation of food proteins. Recovery of

bioactive peptides having inhibitory effects on angioten-

sin-converting enzyme from tryptic hydrolysates of casein

was a pioneering case study; the peptides had a substan-

tially greater affinity (yield � 99%) towards the polymer-

rich phase of PEG/potassium phosphate ATPSs [25]. As a

recent instance, recovery of antioxidant peptides from

hydrolysates of whey proteins was achieved using ATPSs

of poly(ethylene glycol-ran-propylene glycol) monobutyl

ether/phosphate [26] and 1-propanol/NaH2PO4 [27].

We expect that ATPS emulsions can be additionally

exploited for the in situ generation of bioactive peptides

from food proteins and concurrent fractionation of the

peptides within either of the aqueous phases. This appli-

cation would rely on preferential partitioning of food

proteins and proteolytic enzymes within the inner com-

partment and extraction of the released peptides into the

exterior phase. Since the phase behavior can be tuned

Figure 1

PEG-CaCl2bath

PEG inlet

Outlet

Glass slide PDMS

DEX-alginateinlet Intermed

Schematic illustration of the setup used for formation of pollen-like alginate

inside the outlet tubing, and flow into a bath of PEG and CaCl2 for 30 s. Th

droplets proceed over time. Scale bar indicates 50 mm [29�].


extensively, we expect that several possibilities for indus-

trial use of food-grade all-aqueous emulsions for proteol-

ysis reactions are within reach.

Fabrication of microgels using all-aqueous emulsions

The utilization of all-aqueous emulsions for bioparticle

(microgel) synthesis via solidification of the inner phase

is appealing from the perspectives of consumer and

environmental concerns. Synthesis of hydrogel micro-

particles through crosslinking of methacrylated dextran

(mDex) in mDex-in-PEG or dimethacrylated PEG

(m2PEG) in m2PEG-in-Dex (or m2PEG-in-magnesium

sulfate) emulsions were pioneering instances. Similarly,

gelatin microparticles were made by emulsification of a

gelatin solution in an aqueous solution of poly(vinylpyr-

rolidone) or dextran at high temperature [28] and subse-

quent cooling to ambient conditions. In addition to

solidification of the polymer which constitutes the inter-

nal phase, all-aqueous emulsions have been used as

scaffolds for bioparticle synthesis via crosslinking of a

guest ingredient in the dispersed phase. Recently, algi-

nate which mainly partitioned into the inner phase of

Dex-in-PEG emulsions was crosslinked with CaCl2 to

yield pollen-like microparticles (i.e. non-spherical vehi-

cles with protruding spikes). For this, a flow-focusing

microfluidic device (Figure 1) was employed to make

alginate-Dex droplets (50�80 mm) in the PEG phase,

followed by polymerization in a bath containing PEG

and CaCl2. In the bath, a sudden change in concentration

DEX-alginate

PEG

PEG-CaCl2

iate PEGA polymerized DEX particle, 40minutes after reaching the bath

Current Opinion in Food Science

microparticles. The alginate-containing Dex droplets are generated

e polymerization and growth of spikes across the DEX–alginate



across the Dex-PEG interface occurs, resulting in for-

mation of alginate spikes across the droplet surface, of

which the length could be tuned by the PEG concen-

tration [29�].

Comparable to ionic microgelation, which takes place

between oppositely charged ions and biopolymers, elec-

trostatic attraction between two oppositely charged bio-

polymers can be exploited for synthesis of microgel

particles. Bicomponent protein microparticles consisting

of hemoglobin (positively charged) and bovine serum

albumin (BSA, negatively charged) or immunoglobulin

G (negatively charged) were prepared in the droplet

phase of Dex-in-PEG emulsions at pH 7.0 (Figure 2a).

Before emulsification, the anionic and cationic proteins

were placed in the Dex and PEG phases, respectively.

Once the emulsion droplets were fabricated via a glass

capillary-based microfluidic electrospray strategy, BSA

remained in the Dex phase (partitioning coefficient

was 0.96 in Dex and 0.04 in PEG), whereas, hemoglobin

migrated from the PEG phase into the Dex phase (parti-

tioning coefficient in Dex was 0.7). Gradual changes took

place with the Dex droplets becoming darker because of

the electrostatic attractive, as well as hydrophobic inter-

actions between the proteins [30]. Comparable methods

can be used for preparation of bicomponent food protein,

and protein-polysaccharide (positively charged proteins

and negatively charged polysaccharides) composite

microgels. Moreover, bio-microgels consisting of a

charged bioactive cargo of interest can be encapsulated.

The PEG phase needs to be removed for harvesting the

bioparticles.

Interfacial reactions using all-aqueousemulsionsCapsule formation

Besides microgel formation, electrostatic attraction

between charged species can be used for solidification

of the interface, resulting in formation of microcapsules (i.

e. the internal phase remains liquid). The first report [31�]included the self-assembly of oppositely charged poly-

electrolytes, namely poly(allylamine hydrochloride)

(PAH) and poly(sodium-4-styrenesulfonate) (PSS) at

the W-W interface of all-aqueous PEG-Dex emulsions

(Figure 2b). Formation of microcapsules (i.e. on-droplet

self-assembly) rather than microgels (i.e. in-droplet

self-assembly) requires several customizations. A key

point is prevention of free diffusion of polyelectrolytes

across the interface [31�]. As the interfacial tension in

ATPSs is very low, polyelectrolytes are not adsorbed to

the interface, but rather tend to migrate across the inter-

face due to concentration differences between the phases.

It is crucial to tune the relative fluxes of polyelectrolytes

so that they meet at the interface. Moreover, the concen-

tration of polymers in each aqueous phase has to be close

to the equilibrium condition in the ternary phase diagram

of the corresponding ATPS, so that, intermixing of the


polymers is weak when the two solutions come into

contact [32]. In order to tailor polyelectrolyte diffusion,

they were added to the aqueous phase with poorer

affinity. The polyanion PSS with much higher affinity

for Dex (partitioning coefficient 0.69) was added to the

PEG phase and the polycation PAH with almost no

preference between the phases (partitioning coefficient

in Dex: 0.51) was added into the Dex phase. Once the

Dex phase was electrosprayed into the PEG phase, PSS

strongly migrated from the continuous PEG phase

inwards to the droplet phase (Dex), while PAH weakly

migrated outwards. The opposite transfer of PSS and

PAH caused complexation at the W-W interface, thus

forming microcapsules [31�].

Recently, BSA-loaded or catalase-loaded colloidosomes

were fabricated by in situ generation of colloidal particles

at all-aqueous interfaces. Colloidosomes are microcap-

sules with shells consisting of colloidal particles. The

dispersed phase, which consisted of Dex containing

Na-alginate and urease (also BSA or catalase) was peri-

odically injected into a continuous phase of PEG using a

glass capillary microfluidic device (Figure 2c). Upon

existing the microfluidic device, the emulsion droplets

were directed into a bath containing PEG, CaCl2 and

urea: CaCl2 and urea could freely diffuse across the

interface and enter into the Dex phase, while, urease

tended to stay in the Dex phase. The diffusion of Ca2+

crosslinked the alginate at the droplet surface, and urease

concurrently formed ammonium carbonate from urea that

in the presence of Ca2+ ions generated CaCO3 particles.

The CaCO3 particles nucleated and grew into a solid shell

on the microgels (Figure 2c). The encapsulation effi-

ciency was high (>85%) [33].

Reactions at the droplet interface

The W-W interface can be exploited as a liquid-staying

reaction site for reactants partitioned in the discrete

phases of all-aqueous emulsions. To illustrate this, an

enzyme-assisted oxidation reaction was investigated in

macroscopically phase-separated solutions of PEG and

sodium citrate (Figure 3). The citrate-rich phase con-

tained the enzymes glucose oxidase (28:1 concentration

excess compared to the PEG-rich phase) and horseradish

peroxidase (1.6:1), as well as high concentrations of glu-

cose (1.9:1) and peroxide (1.65:1). The hydrophobic

substrate, amplex red and the reaction product, resorufin

strongly partitioned into the PEG-rich phase. First, resor-

ufin formation (pink color) was observed in the citrate-

rich phase, and after 10 min also at the interface.

The reaction product continuously partitioned into the

PEG-rich phase, so that after 3 hour the PEG-rich phase

was uniformly pink [23��]. Fabrication of fibrillosomes

through growing amyloid fibrils from hen egg lysozyme

as monomer at all-aqueous interfaces seeded with pre-

formed lysozyme fibrils also holds a merit [34].



Figure 2

(a)

(b) (c)

a

b c d

Droplet phase

Droplet template

Continuous phase

Polycations Polyanions

PEG, urea

PEG

PEGinlet

Dex, Na-algand ureaseinlet

Dex, Na-algand urease

Urease-mediatedbiomineralization

Colloidosome formed fromgelation and biomineralization

Dex PEG Na-alg Urease Urea Ca-algCa2+ CaCO3

CaCl2

“Drainage”5mg/ml 10mg/ml 15mg/ml 20mg/ml

Concentrations of PSS & FITC-PAH

Microcapsule

Droplet Phase Continuous Phase Hb (+) BSA (-)

Assembly

AffinityPartitioning

Dual -Component ProteinMicroparticles


(a) Schematic illustration of the formation of bicomponent (dual component) protein particles in all-aqueous emulsion (a); Optical microscope

images of hemoglobin–bovine serum albumin microparticles taken at the (b) beginning and (c) end of the fabrication process; Cryo-scanning

electron microscopy image of the microparticles (d). Scale bar: 100 mm [30].

(b) Scheme of the self-assembly of polyelectrolytes at the W-W interface and confocal microscopy images of the microcapsules, scale bar: 50 mm

[31�].(c) Optical images of the microfluidic device (a), the taper showing droplet movement when the pump was turn on (b), and off (c), and Dex

droplets containing Na-alginate moving in the outer tube (d). The bottom scheme illustrates the fabrication mechanism of Ca-alginate microgels

through urease-mediated biomineralization (CaCO3 generation) at the Dex-alginate phase interface [33].

www.sciencedirect.com Current Opinion in Food Science 2020, 33:165–172


Figure 3

PEG-rich phase

PEG-rich phase

Citrate-rich phase

Citrate-rich phase

Citrate-rich phase

1.5 5 10 30 60 90 180Minutes

resorufin

D-glucono-1,5-lactoneD-(+)-glucose

glucoseoxidase

horseradishperoxidase

Amplex Red


Progress of the reaction between amplex red and hydrogen peroxide in the presence of horseradish peroxidase at the W-W interface between a

citrate-rich aqueous phase and a PEG-rich aqueous phase [23��].

Microfluidics for all-aqueous emulsionsMicrofluidic emulsification enables fabrication of highly

monodisperse all-aqueous emulsions. Because of the

inherently laminar flow conditions in the small channels,

the droplet formation is highly repeatable and can be

precisely controlled at low energy input [35,36�]. Appli-

cation of microfluidics for generation of all-aqueous emul-

sions is rapidly developing. Because of the very low

interfacial tension of all aqueous emulsions, the droplet

formation mechanism is rather different from those pre-

sented for other two-phase systems. For example, for oil

in water emulsions, inflow of the water phase at the point

of droplet formation is essential for droplet formation to

take place, and this occurs through the ‘gutters’ that form

at the edges/corners of the rectangular channel [37–39].

For ATPSs, destabilization takes place through shear

forces, which leads to destabilization of the neck which

can perfectly take place in round channels. Development

of non-planar microfluidic channels is ongoing and should

be taken in consideration for future all-aqueous emulsion

production [40].

Recently, a microfluidics platform to make double and

triple all-aqueous emulsions based on hybridization of a


conventional microfluidic flow-focusing geometry with a

coaxial microneedle and a glass capillary embedded in

flow-focusing junctions was presented for making Dex-in-

PEG-in-Dex emulsions [41]. Fabrication of such micro-

fluidic devices, however, is sometimes cumbersome and

not easily available for scientists without a background in

microfabrication. Recent developments in microfluidic

fabrication have brought this out of the clean room, using

3D printers, laser cutters and sacrificial mold to create

microchannels [42–45]. One example is the Embedded

Scaffold RemovinG Open Technology (ESCARGOT), a

simple and versatile 3D printing method which allows to

make multilayered and intricate micrometric channels in

a single block of polydimethylsiloxane (PDMS) [46]. This

can be combined with various components such as heat-

ing units, color sensors and even a solenoidal micro-coil

allowing NMR-spectroscopy in flow [46] for example, for

the quantification of reactions in flow [47].

Due to the small amounts of reactants, analytical detec-

tion and reaction monitoring (on single droplet level) are

usually challenging. Still, some options are available that

use optical detection (mostly fluorescence), electrochem-

ical signals, but also Mass Spectrometry and NMR [48�].



As analytical devices are improving in sensitivity and

specificity year after year, the detection of small-scale

reactions will improve along with the analytical devices.

In-line ultrasound-enhanced Raman spectroscopy for sus-

pensions [49] and in-line nuclear magnetic resonance

(NMR) embedded into flow reactors [50] are instances

of the analytical techniques which can be used for moni-

toring reactions in microfluidic cells.

Concluding remarksAll-aqueous emulsions are a promising tool for running

bioconversion reactions, synthesis of bio-microgels and

bio-microcapsules. The main advantages of using all-

aqueous emulsions as microreaction vessels include

avoiding the utilization of synthetic surfactants, and

organic solvents. Moreover, the emulsion fabrication is

readily achieved on large scale without need to high-

pressure and high-speed homogenizers.

Although the application of all-aqueous emulsions for

microgel synthesis is already advanced, performing reac-

tions at the W-W interfaces is still in its infancy. The W-W

interfacial reactions are promising for making food-grade

core-liquid microcapsules, as well as to produce com-

pounds that leave the interface and partition into either

of the aqueous phases.

Conflict of interest statementNothing declared.

CRediT authorship contribution statementAshkan Madadlou: Conceptualization, Writing - original

draft. Vittorio Saggiomo: Writing - original draft. KarinSchroen: Writing - review & editing. Vincenzo Fogliano:Writing - review & editing.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest�� of outstanding interest

1. Xin Z, Skrydstrup T: Liquid marbles: a promising and versatileplatform for miniaturized chemical reactions. Angew Chem - IntEd 2019, 58:11952-11954.

2. Pera-Titus M, Leclercq L, Clacens J-M, De Campo F, Nardello-Rataj V: Pickering interfacial catalysis for biphasic systems:from emulsion design to green reactions. Angew Chem Int Ed2015, 54:2006-2021.

3. Piradashvili K, Alexandrino EM, Wurm FR, Landfester K:Reactions and polymerizations at the liquid–liquid interface.Chem Rev 2016, 116:2141-2169.

4. Dong Z, Liu Z, Shi J, Tang H, Xiang X, Huang F, Zheng M: Carbonnanoparticle-stabilized pickering emulsion as a sustainableand high-performance interfacial catalysis platform forenzymatic esterification/transesterification. ACS SustainChem Eng 2019, 7:7619-7629.

5. Madadlou A, Jaberipour S, Eskandari MH: Nanoparticulation ofenzymatically cross-linked whey proteins to encapsulatecaffeine via microemulsification/heat gelation procedure. LWT- Food Sci Technol 2014, 57:725-730.


6. Nourbakhsh H, Madadlou A, Emam-Djomeh Z, Wang Y-C,Gunasekaran S: One-pot nanoparticulation of potentiallybioactive peptides and gallic acid encapsulation. Food Chem2016, 210:317-324.

7. Yu D, Xiao S, Tong C, Chen L, Liu X: Dialdehyde starchnanoparticles: preparation and application in drug carrier.Chin Sci Bull 2007, 52:2913-2918.

8. Zou Y, Song J, You X, Yao J, Xie S, Jin M, Wang X, Yan Z, Zhou G,Shui L: Interfacial complexation induced controllablefabrication of stable polyelectrolyte microcapsules using all-aqueous droplet microfluidics for enzyme release. ACS ApplMater Interfaces 2019, 11:21227-21238.

9.�

Dewey DC, Strulson CA, Cacace DN, Bevilacqua PC, Keating CD:Bioreactor droplets from liposome-stabilized all-aqueousemulsions. Nat Commun 2014:5

A strategic review on particle-stabilized and segregatively phase sepa-rated biopolymer mixtures.

10. Dickinson E: Particle-based stabilization of water-in-wateremulsions containing mixed biopolymers. Trends Food SciTechnol 2019, 83:31-40.

11. Iqbal M, Tao Y, Xie S, Zhu Y, Chen D, Wang X, Huang L, Peng D,Sattar A, Shabbir MAB et al.: Aqueous two-phase system(ATPS): an overview and advances in its applications. BiolProced Online 2016, 18:18.

12. Cheng T-L, Chuang K-H, Chen B-M, Roffler SR: Analyticalmeasurement of PEGylated molecules. Bioconjug Chem 2012,23:881-899.

13. Madadlou A, Floury J, Dupont D: Structural assessment andcatalytic oxidation activity of hydrophobized whey proteins. JAgric Food Chem 2018, 66:12025-12033.

14. Madadlou A, Saint-Jalmes A, Guyomarc’h F, Floury J, Dupont D:Development of an aqueous two-phase emulsion usinghydrophobized whey proteins and erythritol. FoodHydrocolloids 2019, 93:351-360.

15. Madadlou A, Famelart M-H, Pezennec S, Rousseau F, Floury J,Dupont D: Interfacial and (emulsion) gel rheology ofhydrophobised whey proteins. Int Dairy J 2020, 100:104556.

16. Shibata C, Iwashita K, Shiraki K: Selective separation method ofaggregates from IgG solution by aqueous two-phase system.Protein Exp Purif 2019, 161:57-62.

17. Silverio SC, Rodrıguez O, Tavares APM, Teixeira JA, Macedo EA:Laccase recovery with aqueous two-phase systems: Enzymepartitioning and stability. J Mol Catal B Enzym 2013, 87:37-43.

18. Weißenborn E, Braunschweig B: Hydroxypropyl cellulose as agreen polymer for thermo-responsive aqueous foams. SoftMatter 2019, 15:2876-2883.

19. Brandelli A, Sala L, Kalil SJ: Microbial enzymes forbioconversion of poultry waste into added-value products.Food Res Int 2015, 73:3-12.

20. Lahore HMF, Miranda MV, Fraile ER, Bonino MJB de J,Cascone O: Partition behaviour and purification of a Mucorbacilliformis acid protease in aqueous two-phase systems.Process Biochem 1995, 30:615-621.

21. Reh G, Spelzini D, Tubio G, Pico G, Farruggia B: Partition featuresand renaturation enhancement of chymosin in aqueous two-phase systems. J Chromatogr B 2007, 860:98-105.

22. Andersson E, Hahn-Hagerdal B: Bioconversions in aqueoustwo-phase systems. Enzyme Microb Technol 1990, 12:242-254.

23.��

Aumiller WM, Davis BW, Hashemian N, Maranas C, Armaou A,Keating CD: Coupled enzyme reactions performed inheterogeneous reaction media: experiments and modeling forglucose oxidase and horseradish peroxidase in a PEG/citrateaqueous two-phase system. J Phys Chem B 2014, 118:2506-2517

A pioneering and unique report on accomploishing enzymatic/chemicalreactions at the water-water interface that stays liquid.

24. Badhwar P, Kumar P, Dubey KK: Extractive fermentation forprocess integration and amplified pullulan production by A.pullulans in aqueous two phase systems. Sci Rep 2019, 9:32.


http://refhub.elsevier.com/S2214-7993(20)30052-7/sbref0005





















































































25. de Souza EC, Dos Reis Coimbra JS, de Oliveira EB, Bonomo RCF:Recovery of casein-derived peptides with in vitro inhibitoryactivity of angiotensin converting enzyme (ACE) usingaqueous two-phase systems. J Chromatogr B 2014, 973:84-88.

26. Jiang B, Zhang X, Yuan Y, Qu Y, Feng Z: Separation ofantioxidant peptides from pepsin hydrolysate of whey proteinisolate by ATPS of EOPO co-polymer (UCON)/phosphate. SciRep 2017, 7:13320.

27. Jiang B, Na J, Wang L, Li D, Liu C, Feng Z: Separation andenrichment of antioxidant peptides from whey protein isolatehydrolysate by aqueous two-phase extraction and aqueoustwo-phase flotation. Foods 2019, 8:34.

28. Franssen O, Hennink WE: A novel preparation method forpolymeric microparticles without the use of organic solvents.Int J Pharm 1998, 168:1-7.

29.�

Abbasi N, Navi M, Nunes JK, Tsai SSH: Controlled generation ofspiky microparticles by ionic cross-linking within an aqueoustwo-phase system. Soft Matter 2019, 15:3301-3306

A unique and facile chemical synthesis approach, for controlled genera-tion of pollen-like microparticles, based on ionic cross-linking of alginateand calcium chloride (CaCl2), within an all-biocompatible ATPS of DEXand PEG is developed.

30. Deng Y, Ma Q, Yuan H, Lum GC, Shum HC: Development of dual-component protein microparticles in all-aqueous systems forbiomedical applications. J Mater Chem B 2019, 7:3059-3065.

31.�

Ma Q, Song Y, Kim JW, Choi HS, Shum HC: Affinity partitioning-induced self-assembly in aqueous two-phase systems:templating for polyelectrolyte microcapsules. ACS Macro Lett2016, 5:666-670

The affinity partitioning of polyelectrolytes in ATPS to induce their self-assembly into polyelectrolyte microcapsules is introduced.

32. Hann SD, Niepa THR, Stebe KJ, Lee D: One-step generation ofcell-encapsulating compartments via polyelectrolytecomplexation in an aqueous two phase system. ACS ApplMater Interfaces 2016, 8:25603-25611.

33. Qu F, Meng T, Dong Y, Sun H, Tang Q, Liu T, Wang Y: Aqueoustwo-phase droplet-templated colloidosomes composed ofself-formed particles via spatial confined biomineralization.ACS Appl Mater Interfaces 2019, 11:35613-35621.

34. Song Y, Shimanovich U, Michaels TCT, Ma Q, Li J, Knowles TPJ,Shum HC: Fabrication of fibrillosomes from droplets stabilizedby protein nanofibrils at all-aqueous interfaces. Nat Commun2016, 7:12934.

35. Muijlwijk K, Berton-Carabin C, Schroen K: Cross-flowmicrofluidic emulsification from a food perspective. TrendsFood Sci Technol 2016, 49:51-63.

36.�

Schroen K, Bliznyuk O, Muijlwijk K, Sahin S, Berton-Carabin CC:Microfluidic emulsification devices: from micrometer insightsto large-scale food emulsion production. Curr Opin Food Sci2015, 3:33-40

The paper addresses microfluidic techniques for emulsion preparation.Both scale-up of standard microfluidic devices, and characterisation ofemulsion formation and stability using purpose built microfluidics arediscussed.

37. ten Klooster S, Sahin S, Schroen K: Monodisperse dropletformation by spontaneous and interaction based mechanismsin partitioned EDGE microfluidic device. Sci Rep 2019, 9:7820.

38. van der Graaf S, Steegmans MLJ, van der Sman RGM,Schroen CGPH, Boom RM: Droplet formation in a T-shapedmicrochannel junction: a model system for membraneemulsification. Colloids Surf A Physicochem Eng Asp 2005,266:106-116.


39. Garstecki P, Fuerstman MJ, Stone HA, Whitesides GM: Formationof droplets and bubbles in a microfluidic T-junction—scalingand mechanism of break-up. Lab Chip 2006, 6:437.

40. Hwang Y, Candler RN: Non-planar PDMS microfluidic channelsand actuators: a review. Lab Chip 2017, 17:3948-3959.

41. Jeyhani M, Thevakumaran R, Abbasi N, Hwang DK, Tsai SSH:Microfluidic generation of all-aqueous double and tripleemulsions. Small 2020, 16:1906565.

42. Goh WH, Hashimoto M: Fabrication of 3D microfluidic channelsand in-channel features using 3D printed, water-solublesacrificial mold. Macromol Mater Eng 2018, 303:1700484.

43. Dahlberg T, Stangner T, Zhang H, Wiklund K, Lundberg P,Edman L: Andersson M: 3D printed water-soluble scaffolds forrapid production of PDMS micro-fluidic flow chambers. SciRep 2018, 8:3372.

44. Chen YY, Kingston BR, Chan WCW: Transcribing in vivo bloodvessel networks into in vitro perfusable microfluidic devices.Adv Mater Technol 2020, 5:2000103.

45. Walsh DI, Kong DS, Murthy SK, Carr PA: Enabling microfluidics:from clean rooms to makerspaces. Trends Biotechnol 2017,35:383-392.

46. Saggiomo V, Velders AH: Simple 3D printed scaffold-removalmethod for the fabrication of intricate microfluidic devices.Adv Sci 2015, 2:1500125.

47. Mompean M, Sanchez-Donoso RM, de la Hoz A, Saggiomo V,Velders AH, Gomez MV: Pushing nuclear magnetic resonancesensitivity limits with microfluidics and photo-chemicallyinduced dynamic nuclear polarization. Nat Commun 2018, 9:108.

48.�

Suea-Ngam A, Howes PD, Srisa-Art M, DeMello AJ: Dropletmicrofluidics: from proof-of-concept to real-world utility?Chem Commun 2019, 55:9895-9903

A recent review on droplet microfluidics, from fabrication to analysis andfuture applications.

49. Wieland K, Tauber S, Gasser C, Rettenbacher LA, Lux L, Radel S,Lendl B: In-line ultrasound-enhanced Raman spectroscopyallows for highly sensitive analysis with improved selectivity insuspensions. Anal Chem 2019, 91:14231-14238.

50. Giraudeau P, Felpin F-X: Flow reactors integrated with in-linemonitoring using benchtop NMR spectroscopy. React ChemEng 2018, 3:399-413.

51. Agrawal K, Verma P: Potential removal of hazardous wastesusing white laccase purified by ATPS–PEG–salt system: anoperational study. Environ Technol Innov 2020, 17:100556.

52. Simental-Martınez J, Montalvo-Hernandez B, Rito-Palomares M,Benavides J: Application of aqueous two-phase systems forthe recovery of bioactive low-molecular weight compounds.Sep Sci Technol 2014, 49:1872-1882.

53. Wang L, Li W, Liu Y, Zhi W, Han J, Wang Y, Ni L: Green separationof bromelain in food sample with high retention of enzymeactivity using recyclable aqueous two-phase systemcontaining a new synthesized thermo-responsive copolymerand salt. Food Chem 2019, 282:48-57.

54. Veloso AV, Silva BC, Bomfim SA, de Souza RL, Soares CMF,Lima AS: Selective and continuous recovery of ascorbic acidand vanillin from commercial diet pudding waste using anaqueous two-phase system. Food Bioprod Process 2020,119:268-276.













































































































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